143 results on '"Marcus A. Hemminga"'
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2. Speeding Up a Genetic Algorithm for EPR-Based Spin Label Characterization of Biosystem Complexity.
- Author
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Aleh A. Kavalenka, Bogdan Filipic, Marcus A. Hemminga, and Janez Strancar
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- 2005
- Full Text
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3. Exploring the Local Conformational Space of a Membrane Protein by Site-Directed Spin Labeling.
- Author
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David Stopar, Janez Strancar, Ruud B. Spruijt, and Marcus A. Hemminga
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- 2005
- Full Text
- View/download PDF
4. Spin Label EPR-Based Characterization of Biosystem Complexity.
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Janez Strancar, Tilen Koklic, Zoran Arsov, Bogdan Filipic, David Stopar, and Marcus A. Hemminga
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- 2005
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5. Artificial Neural Network Modification of Simulation-Based Fitting: Application to a Protein-Lipid System.
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Petr V. Nazarov, Vladimir V. Apanasovich, Vladimir M. Lutkovski, Mikalai M. Yatskou, Rob B. M. Koehorst, and Marcus A. Hemminga
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- 2004
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6. Distance Measurements on Orthogonally Spin-Labeled Membrane Spanning WALP23 Polypeptides
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Marcus A. Hemminga, Maxim Yulikov, Petra Lueders, Gunnar Jeschke, and Heidrun Jäger
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spectroscopy ,Nitroxide mediated radical polymerization ,Biophysics ,Analytical chemistry ,Gadolinium ,010402 general chemistry ,01 natural sciences ,Ion ,law.invention ,Paramagnetism ,field pulsed epr ,trichogin ga-iv ,law ,Materials Chemistry ,islet amyloid polypeptide ,Physical and Theoretical Chemistry ,Spin (physics) ,Spectroscopy ,Electron paramagnetic resonance ,010405 organic chemistry ,Chemistry ,alpha-helical peptides ,gd3+ complexes ,phospholipid membrane ,Electron Spin Resonance Spectroscopy ,hydrophobic mismatch ,dipole-dipole interactions ,electron double-resonance ,0104 chemical sciences ,Surfaces, Coatings and Films ,Biofysica ,Membrane ,Nitrogen Oxides ,Spin Labels ,Peptides ,Selectivity - Abstract
EPR-based Gd(III)-nitroxide distance measurements were performed on a series of membrane-incorporated orthogonally labeled WALP23 polypeptides. The obtained distance distributions were stable upon the change of detection frequency from 10 GHz (X-band) to 35 GHz (Q-band). The alpha-helical pitch of WALP23 polypeptide could be experimentally observed, despite the flexibility of the two spin labels. The spectroscopic properties of Gd(III) ions and nitroxide radicals allow detecting both types of paramagnetic species selectively in different EPR experiments. In particular, this spectroscopic selectivity allows for supplementing Gd(III)-nitroxide distance measurements with independent checks of polypeptide aggregation and with measurements of the local environment of the nitroxide spin labels. All mentioned additional checks do not require preparation of further samples, as it is the case in the experiments with pairs of identical nitroxide spin labels.
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- 2013
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7. Distance determination from dysprosium induced relaxation enhancement: a case study on membrane-inserted WALP23 polypeptides
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Petra Lueders, Maxim Yulikov, Gunnar Jeschke, Marcus A. Hemminga, Heidrun Jäger, Sahand Razzaghi, and René Tschaggelar
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Nitroxide mediated radical polymerization ,Distance measurements ,Radical ,Analytical chemistry ,Biophysics ,chemistry.chemical_element ,selection ,dipolar spectroscopy ,010402 general chemistry ,01 natural sciences ,EPR/ESR ,biomacromolecules ,law.invention ,electron-paramagnetic-resonance ,law ,Inversion recovery ,Physical and Theoretical Chemistry ,spin-lattice-relaxation ,Electron paramagnetic resonance ,Molecular Biology ,Lanthanide ions ,Biomacromolecules ,Dipolar spectroscopy ,010405 organic chemistry ,Chemistry ,pulsed dipolar esr ,Relaxation (NMR) ,Spin–lattice relaxation ,Resonance ,Condensed Matter Physics ,0104 chemical sciences ,labels ,Membrane ,Biofysica ,ddc:540 ,range ,Dysprosium ,epr spectroscopy ,nitroxide ,performance - Abstract
Molecular Physics, 111 (18-19), ISSN:0026-8976, ISSN:1362-3028
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- 2013
8. Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles
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Marcus A. Hemminga, Rob B. M. Koehorst, Afonso M.S. Duarte, and Edwin R. de Jong
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Circular dichroism ,Vacuolar Proton-Translocating ATPases ,Stereochemistry ,Molecular Sequence Data ,Biophysics ,Peptide ,V-ATPase ,Saccharomyces cerevisiae ,Fluorescence spectroscopy ,Protein Structure, Secondary ,5th transmembrane segment ,nmr ,Residue (chemistry) ,Aggregation ,mimicking ,detergent ,Secondary structure ,Membrane-mimicking solvent ,Amino Acid Sequence ,Peptide sequence ,Protein secondary structure ,major coat protein ,Spectroscopy ,Micelles ,chemistry.chemical_classification ,proton translocation channel ,Original Paper ,EPS-2 ,Protein Stability ,Circular Dichroism ,Cell Membrane ,Tryptophan ,Sodium Dodecyl Sulfate ,General Medicine ,bacteriophage-m13 ,dodecyl-sulfate micelles ,Transmembrane protein ,Peptide Fragments ,Biofysica ,Spectrometry, Fluorescence ,chemistry ,Biochemistry ,Solubility ,membrane-proteins ,escherichia-coli ,SDS micelles ,Protein Binding - Abstract
The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an alpha-helical conformation or aggregated beta-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to beta-sheet formation and subsequent aggregation.
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- 2009
9. Site-Directed Spin-Labeling Study of the Light-Harvesting Complex CP29
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Ruud B. Spruijt, Aleh Kavalenka, Roberta Croce, Cor J. A. M. Wolfs, Marcus A. Hemminga, Janez Štrancar, Herbert van Amerongen, and Electron Microscopy
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CONFORMATIONAL-CHANGES ,photosystem-ii subunit ,Protein Conformation ,Arabidopsis ,Light-Harvesting Protein Complexes ,law.invention ,Light-harvesting complex ,energy-transfer ,BIOSYSTEM COMPLEXITY ,Protein structure ,Spinacia oleracea ,law ,Electron paramagnetic resonance ,Spin label ,CHLOROPHYLL-A/B PROTEIN ,PLANT ANTENNA PROTEIN ,MEMBRANE-PROTEINS ,Chemistry ,EPS-3 ,Membrane ,Transmembrane protein ,Transmembrane domain ,Biofysica ,ESCHERICHIA-COLI ,Protein body ,ENERGY-TRANSFER ,PHOTOSYSTEM-II SUBUNIT ,Biophysics ,binding protein ,BINDING PROTEIN ,Models, Biological ,conformational-changes ,Escherichia coli ,Computer Simulation ,n-terminal domain ,N-TERMINAL DOMAIN ,plant antenna protein ,Arabidopsis Proteins ,Electron Spin Resonance Spectroscopy ,Photosystem II Protein Complex ,Site-directed spin labeling ,Carotenoids ,Crystallography ,Mutation ,membrane-proteins ,Mutagenesis, Site-Directed ,escherichia-coli ,chlorophyll-a/b protein ,Apoproteins ,biosystem complexity - Abstract
The topology of the long N-terminal domain (similar to 100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.
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- 2009
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10. From 'I' to 'L' and back again: the odyssey of membrane-bound M13 protein
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Ruud B. Spruijt, Petr V. Nazarov, Rob B. M. Koehorst, Werner L. Vos, and Marcus A. Hemminga
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Coat ,fd ,helix ,Protein Conformation ,Membrane bound ,viruses ,Molecular Sequence Data ,domain ,Biophysics ,amino-acids ,Coat protein ,Biology ,Models, Biological ,Biochemistry ,nmr-spectroscopy ,Amino Acid Sequence ,Molecular Biology ,major coat protein ,environments ,EPS-2 ,filamentous bacteriophages ,Membrane Proteins ,resolution ,dynamics ,biology.organism_classification ,Biofysica ,Membrane protein ,Filamentous bacteriophage ,Capsid Proteins ,Bacteriophage M13 - Abstract
The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein. Recently, site-directed labelling approaches have enabled the study of the coat protein under conditions that more closely mimic the in vivo membrane-bound state. Interestingly, the structure that has emerged from this work is I-shaped and similar to the structure in the phage-bound state.
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- 2009
11. An N-terminal diacidic motif is required for trafficking of the maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the plasma membrane
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Marcus A. Hemminga, Jan Willem Borst, François Chaumont, Urszula Miecielica, and Enric Zelazny
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Gene isoform ,polarized trafficking ,Mutant ,Amino Acid Motifs ,Molecular Sequence Data ,Biophysics ,Aquaporin ,Biochemie ,Plant Science ,Biology ,Aquaporins ,Endoplasmic Reticulum ,Zea mays ,Biochemistry ,surface expression ,quality-control ,subcellular-localization ,Gene Expression Regulation, Plant ,Genetics ,Protein Isoforms ,Amino Acid Sequence ,Plant Proteins ,EPS-2 ,Endoplasmic reticulum ,Cell Membrane ,endoplasmic-reticulum ,ER retention ,plant-cells ,Cell Biology ,water channel ,Plant cell ,Subcellular localization ,acidic sequence ,Fusion protein ,Cell biology ,arginine methylation ,Protein Transport ,Biofysica ,protein ,Sequence Alignment - Abstract
Maize plasma membrane aquaporins (ZmPIPs, where PIP is the plasma membrane intrinsic protein) fall into two groups, ZmPIP1s and ZmPIP2s, which, when expressed alone in mesophyll protoplasts, are found in different subcellular locations. Whereas ZmPIP1s are retained in the endoplasmic reticulum (ER), ZmPIP2s are found in the plasma membrane (PM). We previously showed that, when co-expressed with ZmPIP2s, ZmPIP1s are relocalized to the PM, and that this relocalization results from the formation of hetero-oligomers between ZmPIP1s and ZmPIP2s. To determine the domains responsible for the ER retention and PM localization, respectively, of ZmPIP1s and ZmPIP2s, truncated and mutated ZmPIPs were generated, together with chimeric proteins created by swapping the N- or C-terminal regions of ZmPIP2s and ZmPIP1s. These mutated proteins were fused to the mYFP and/or mCFP, and the fusion proteins were expressed in maize mesophyll protoplasts, and were then localized by microscopy. This allowed us to identify a diacidic motif, DIE (Asp-Ile-Glu), at position 4-6 of the N-terminus of ZmPIP2;5, that is essential for ER export. This motif was conserved and functional in ZmPIP2;4, but was absent in ZmPIP2;1. In addition, we showed that the N-terminus of ZmPIP2;5 was not sufficient to cause the export of ZmPIP1;2 from the ER. A study of ZmPIP1;2 mutants suggested that the N- and C-termini of this protein are probably not involved in ER retention. Together, these results show that the trafficking of maize PM aquaporins is differentially regulated depending on the isoform, and involves a specific signal and mechanism.
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- 2009
12. Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment
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Rob B. M. Koehorst, Ruud B. Spruijt, and Marcus A. Hemminga
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Models, Molecular ,phospholipid-bilayers ,Stereochemistry ,Protein Conformation ,Lipid Bilayers ,water ,Biophysics ,Molecular Probe Techniques ,charge-transfer fluorescence ,chemistry.chemical_compound ,Protein structure ,2-Naphthylamine ,solvent relaxation ,excited-state ,Computer Simulation ,Lipid bilayer ,major coat protein ,Crystallography ,Membranes ,EPS-2 ,Bilayer ,Membrane Proteins ,transmembrane domain ,Fluorescence ,laurdan fluorescence ,Membrane ,Spectrometry, Fluorescence ,Biofysica ,chemistry ,Membrane protein ,Models, Chemical ,Protein topology ,bacteriophage m13 ,Laurdan ,prodan - Abstract
The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly α-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.
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- 2008
13. Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase
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Carlo P. M. van Mierlo, Nico A. J. van Nuland, Marcus A. Hemminga, Cor J. A. M. Wolfs, Michael A. Harrison, Afonso M.S. Duarte, and John B. C. Findlay
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Vacuolar Proton-Translocating ATPases ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Protein subunit ,Molecular Sequence Data ,Biophysics ,Biochemie ,Saccharomyces cerevisiae ,Biochemistry ,sarcoplasmic-reticulum ca2+-atpase ,m13 coat protein ,Protein structure ,nmr-spectroscopy ,V-ATPase ,Amino Acid Sequence ,Peptide sequence ,Protein secondary structure ,Micelles ,EPS-2 ,Chemistry ,Circular Dichroism ,EPS-3 ,circular-dichroism spectra ,V-ATPase subunit a ,v-atpase ,vacuolar (h+)-atpases ,Cell Biology ,NMR ,Peptide Fragments ,Transmembrane protein ,CD ,sodium dodecyl-sulfate ,Transmembrane domain ,Biofysica ,nuclear-magnetic-resonance ,Peptide ,membrane-proteins ,protein secondary structure ,Protons ,Heteronuclear single quantum coherence spectroscopy - Abstract
Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.
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- 2007
14. FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein
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Werner L. Vos, Marcus A. Hemminga, Vladimir V. Apanasovich, Petr V. Nazarov, and Rob B. M. Koehorst
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conformation ,spectroscopy ,micelles ,Lipid Bilayers ,Protein domain ,Biophysics ,Models, Biological ,symbols.namesake ,Protein structure ,energy-transfer ,Naphthalenesulfonates ,Stokes shift ,Fluorescence Resonance Energy Transfer ,Computer Simulation ,Cysteine ,Lipid bilayer ,Fluorescent Dyes ,Membranes ,EPS-1 ,Chemistry ,Vesicle ,Membrane Proteins ,Phosphatidylglycerols ,dynamics ,transmembrane domain ,Fluorescence ,Protein Structure, Tertiary ,Transmembrane domain ,Crystallography ,modulation ,Förster resonance energy transfer ,Biofysica ,Mutagenesis, Site-Directed ,Phosphatidylcholines ,symbols ,peptides ,Capsid Proteins ,fluorescence ,bacteriophage m13 - Abstract
A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/ DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall a-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage. To resolve this problem we have produced several cysteine mutants in the N-terminal domain of the protein and spe- cifically labeled them with the fluorescence probe AEDANS. Analysis of the energy transfer data from the natural Trp-26 to AEDANS using a two-helix protein model and the application of the polarity Stokes shift of the AEDANS fluorescence maxima results in a low-resolution structure of the entire pro- tein, including the tilt and orientation of the N-terminal do- main with respect to the transmembrane domain.
- Published
- 2007
15. Motional Restrictions of Membrane Proteins: A Site-Directed Spin Labeling Study
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Marcus A. Hemminga, Janez Štrancar, David Stopar, and Ruud B. Spruijt
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Models, Molecular ,Circular dichroism ,Protein Conformation ,Biophysics ,Motion ,Structure-Activity Relationship ,residues ,Hydrophobic mismatch ,Protein structure ,Nuclear magnetic resonance ,biological-membranes ,Computer Simulation ,epr ,Spin label ,Lipid bilayer ,major coat protein ,Membranes ,EPS-2 ,Chemistry ,Circular Dichroism ,Electron Spin Resonance Spectroscopy ,lipid interactions ,Biological membrane ,dynamics ,transmembrane domain ,Site-directed spin labeling ,A-site ,Biofysica ,Amino Acid Substitution ,Models, Chemical ,Mutagenesis, Site-Directed ,systems ,Capsid Proteins ,Spin Labels ,bacteriophage m13 ,biosystem complexity - Abstract
Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch.
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- 2006
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16. Anchoring mechanisms of membrane-associated M13 major coat protein
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Ruud B. Spruijt, Marcus A. Hemminga, and David Stopar
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Vesicle-associated membrane protein 8 ,Protein Conformation ,Lipid Bilayers ,Biophysics ,backbone dynamics ,lipid-bilayers ,Biochemistry ,Protein Structure, Secondary ,solid-state ,Residue (chemistry) ,nmr-spectroscopy ,Amino Acids ,Lipid bilayer ,Molecular Biology ,Hydrophobicity scales ,model ,Chemistry ,EPS-2 ,tryptophan residues ,Organic Chemistry ,Peripheral membrane protein ,Cell Membrane ,Cell Biology ,aromatic residues ,transmembrane domain ,Transmembrane protein ,water interface ,Transmembrane domain ,Biofysica ,Membrane protein ,Tyrosine ,Capsid Proteins ,bacteriophage m13 ,Hydrophobic and Hydrophilic Interactions - Abstract
Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer.
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- 2006
17. Membrane-bound Conformation of M13 Major Coat Protein
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Ruud B. Spruijt, Rob B. M. Koehorst, Marcus A. Hemminga, and Werner L. Vos
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Vesicle ,Cell Biology ,computer.file_format ,Biology ,Protein Data Bank ,Biochemistry ,Protein tertiary structure ,Crystallography ,Residue (chemistry) ,Förster resonance energy transfer ,Membrane protein ,Biophysics ,Lipid bilayer ,Molecular Biology ,computer ,Cysteine - Abstract
M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Forster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight α-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.
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- 2005
18. Exploring the Local Conformational Space of a Membrane Protein by Site-Directed Spin Labeling
- Author
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Ruud B. Spruijt, Marcus A. Hemminga, David Stopar, and Janez Strancar
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Models, Molecular ,spectroscopy ,Nitroxide mediated radical polymerization ,m13 ,Molecular model ,Protein Conformation ,micelles ,General Chemical Engineering ,Molecular Conformation ,Biophysics ,Library and Information Sciences ,cytoplasmic membrane ,law.invention ,law ,Amino Acids ,epr ,Electron paramagnetic resonance ,Lipid bilayer ,Spin label ,major coat protein ,Models, Statistical ,EPS-2 ,lipid bilayers ,Chemistry ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,bacteriophage-m13 ,dynamics ,General Chemistry ,Site-directed spin labeling ,Computer Science Applications ,Oxygen ,Solutions ,Crystallography ,Biofysica ,Membrane ,Membrane protein ,Phosphatidylcholines ,escherichia-coli ,Spin Labels ,Algorithms - Abstract
Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the conformational space of the spin-labeled segment of the protein in a membrane system. The method is applied to spin-labeled bacteriophage M13 major coat protein, which is used as a model membrane protein. Single cysteine mutants of the coat protein were labeled with nitroxide spin labels and incorporated in 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) bilayers. The new computational method allows us to monitor distributions of local spatial constraints and molecular mobility, in addition to information about the location of the protein in a membrane. Furthermore, the results suggest that different local conformations may coexist in the membrane protein. The knowledge of different local conformations may help us to better understand the function - structure relationship of membrane proteins.
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- 2005
19. Spin Label EPR-Based Characterization of Biosystem Complexity
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David Stopar, Janez Štrancar, Bogdan Filipič, Zoran Arsov, Marcus A. Hemminga, and Tilen Koklic
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General Chemical Engineering ,domain ,Biophysics ,Structure (category theory) ,Analytical chemistry ,Library and Information Sciences ,law.invention ,law ,motion ,Computer Simulation ,Electron paramagnetic resonance ,Spin label ,major coat protein ,parameters ,model ,Degree (graph theory) ,EPS-2 ,Chemistry ,Cell Membrane ,Condensation ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,lipid interactions ,dynamics ,General Chemistry ,Computer Science Applications ,Characterization (materials science) ,Biofysica ,membranes ,Spin Labels ,paramagnetic-resonance-spectra ,simulations ,Condensation algorithm ,Biological system ,Algorithms - Abstract
Following the widely spread EPR spin-label applications for biosystem characterization, a novel approach is proposed for EPR-based characterization of biosystem complexity. Hereto a computational method based on a hybrid evolutionary optimization (HEO) is introduced. The enormous volume of information obtained from multiple HEO runs is reduced with a novel so-called GHOST condensation method for automatic detection of the degree of system complexity through the construction of two-dimensional solution distributions. The GHOST method shows the ability of automatic quantitative characterization of groups of solutions, e.g. the determination of average spectral parameters and group contributions. The application of the GHOST condensation algorithm is demonstrated on four synthetic examples of different complexity and applied to two physiologically relevant examples − the determination of domains in biomembranes (lateral heterogeneity) and the study of the low-resolution structure of membrane proteins.
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- 2005
20. Membrane Assembly of M13 Major Coat Protein: Evidence for a Structural Adaptation in the Hinge Region and a Tilted Transmembrane Domain
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Cor J. A. M. Wolfs, Marcus A. Hemminga, and Ruud B. Spruijt
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spectroscopy ,fd ,phospholipid-bilayers ,Protein Conformation ,Lipid Bilayers ,Molecular Sequence Data ,Biophysics ,Phospholipid ,cytoplasmic membrane ,Biochemistry ,Protein Structure, Secondary ,residues ,Hydrophobic mismatch ,chemistry.chemical_compound ,Naphthalenesulfonates ,Amino Acid Sequence ,Cysteine ,Lipid bilayer ,Integral membrane protein ,Phospholipids ,environments ,Fluorescent Dyes ,EPS-2 ,Chemistry ,Circular Dichroism ,Virus Assembly ,Sulfhydryl Reagents ,Membrane Proteins ,hydrophobic mismatch ,lipid-bilayer ,Transmembrane protein ,Protein Structure, Tertiary ,Transmembrane domain ,Crystallography ,Spectrometry, Fluorescence ,Biofysica ,Membrane ,Helix ,Mutagenesis, Site-Directed ,escherichia-coli ,Capsid Proteins ,bacteriophage m13 - Abstract
New insights into the low-resolution Structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process.
- Published
- 2004
21. Lipid Bilayer Topology of the Transmembrane α-Helix of M13 Major Coat Protein and Bilayer Polarity Profile by Site-Directed Fluorescence Spectroscopy
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Frank J. Vergeldt, Marcus A. Hemminga, Rob B. M. Koehorst, and Ruud B. Spruijt
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Protein Conformation ,Lipid Bilayers ,domain ,membrane-protein ,Biophysics ,Phospholipid ,Biophysical Theory and Modeling ,Topology ,Protein Structure, Secondary ,residues ,chemistry.chemical_compound ,Protein structure ,Cysteine ,Lipid bilayer phase behavior ,Lipid bilayer ,model ,EPS-2 ,Bilayer ,Phosphatidylglycerols ,dependence ,Lipid bilayer mechanics ,Transmembrane protein ,Protein Structure, Tertiary ,Transmembrane domain ,Crystallography ,Spectrometry, Fluorescence ,Biofysica ,chemistry ,Mutation ,Mutagenesis, Site-Directed ,Phosphatidylcholines ,peptides ,Capsid Proteins ,bacteriophage m13 - Abstract
This article presents a new formalism to perform a quantitative fluorescence analysis using the Stokes shift of AEDANS-labeled cysteine mutants of M13 major coat protein incorporated in lipid bilayers. This site-directed fluorescence spectroscopy approach enables us to obtain the topology of the bilayer-embedded transmembrane alpha-helix from the orientation and tilt angles, and relative bilayer location. Both in pure dioleoylphosphatidylcholine and dioleoylphosphatidylcholine/ dioleoylphosphatidylglycerol ( 4: 1 mol/mol) bilayers, which have a similar bilayer thickness, the tilt angle of the transmembrane helix of the coat protein turns out to be 23degrees +/- 4. Upon decreasing the hydrophobic thickness on going from dieicosenoyl-phosphatidylcholine to dimyristoylphosphatidylcholine, the tilt angle and orientation angle of the transmembrane alpha-helix change. The protein responds to an increase of hydrophobic stress by increasing the tilt angle so as to keep much of its hydrophobic part inside the bilayer. At the same time, the transmembrane helix rotates at its long axis so as to optimize the hydrophobic and electrostatic interactions of the C-terminal phenylalanines and lysines, respectively. The increase of tilt angle cannot completely keep the hydrophobic protein section within the bilayer, but the C-terminal part remains anchored at the acylchain/ glycerol backbone interface at the cost of the N-terminal section. In addition, our analysis results in the pro. le of the dielectric constant of the hydrophobic domain of the bilayer. For all phospholipid bilayers studied the pro. le has a concave shape, with a value of the dielectric constant of 4.0 in the center of the bilayer. The dielectric constant increases on approaching the headgroup region with a value of 12.4 at the acyl-chain/glycerol backbone interface for the various phosphatidylcholines with different chain lengths. For dioleoylphosphatidylcholine/ dioleoylphosphatidylglycerol ( 4: 1 mol/mol) bilayers the value of the dielectric constant at the acyl-chain/glycerol backbone interface is 18.6. In conclusion, the consistency of our analysis shows that the applied cysteine-scanning mutagenesis method with AEDANS labeling of a helical transmembrane protein in combination with a quantitative formalism offers a reliable description of the lipid bilayer topology of the protein and bilayer properties. This also indicates that the spacer link between the protein and AEDANS label is long enough to monitor the local polarity of the lipid environment and not that of the amino-acid residues of the protein, and short enough to have the topology of the protein imposing on the fluorescence properties of the AEDANS label.
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- 2004
22. Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein
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Ruud B. Spruijt, Manuel Prieto, Fábio Fernandes, Luís M. S. Loura, Marcus A. Hemminga, Alexander Fedorov, and Rob B. M. Koehorst
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resonance energy-transfer ,model membranes ,transbilayer distribution ,Biophysics ,law.invention ,Bimolecular fluorescence complementation ,Hydrophobic mismatch ,law ,biological-membranes ,Fluorescence Resonance Energy Transfer ,Electron paramagnetic resonance ,Fluorescent Dyes ,Liposome ,Chromatography ,Membranes ,Chemistry ,EPS-2 ,two-dimensional systems ,electron-spin-resonance ,Biological membrane ,Fluorescence ,Lipids ,Förster resonance energy transfer ,Biofysica ,Liposomes ,escherichia-coli ,Phosphatidylcholines ,Capsid Proteins ,lipids (amino acids, peptides, and proteins) ,fluorescence ,bacteriophage m13 ,Selectivity ,Hydrophobic and Hydrophilic Interactions ,Algorithms ,bilayers - Abstract
Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4′iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies.
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- 2004
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23. Structural characterization of bacteriophage M13 solubilization by amphiphiles
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Ruud B. Spruijt, Cor J. A. M. Wolfs, Marcus A. Hemminga, and David Stopar
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Spin label ,Circular dichroism ,Detergent ,viruses ,Biophysics ,Sodium octyl sulfate ,Biochemistry ,Bacteriophage ,Hydrophobic effect ,Surface-Active Agents ,chemistry.chemical_compound ,Capsid ,Structural Biology ,Electron spin resonance spectroscopy ,Amphiphile ,Sodium dodecyl sulfate ,Conformation ,Molecular Biology ,Protein secondary structure ,Coat protein ,biology ,Circular Dichroism ,Circular dichroism spectroscopy ,biology.organism_classification ,Phage disassembly ,Solutions ,Biofysica ,chemistry ,Critical micelle concentration ,Spin Labels ,EPS ,Sodium decyl sulfate ,Bacteriophage M13 - Abstract
The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle. These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle. The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase. Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure. With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration. A further increase in amphiphile concentration results in massive phage disruption. Phage disruption by amphiphiles appears to be dependent on the phage coat mutations. It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part.
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- 2002
24. Membrane-Anchoring Interactions of M13 Major Coat Protein
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Ruud B. Spruijt, Alexander B. Meijer, Marcus A. Hemminga, and Cor J. A. M. Wolfs
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Lipid Bilayers ,Molecular Sequence Data ,Mutant ,Biophysics ,Biochemistry ,chemistry.chemical_compound ,Hydrophobic mismatch ,Residue (chemistry) ,Capsid ,Naphthalenesulfonates ,Phosphatidylcholine ,Life Science ,Amino Acid Sequence ,Cysteine ,Fluorescent Dyes ,Alanine ,Electron Spin Resonance Spectroscopy ,Tryptophan ,Membrane Proteins ,Transmembrane domain ,Biofysica ,chemistry ,Mutagenesis, Site-Directed ,Phosphatidylcholines ,Capsid Proteins ,Spin Labels ,EPS ,Dimyristoylphosphatidylcholine ,Bacteriophage M13 - Abstract
The response to hydrophobic mismatch of membrane-bound M13 major coat protein is measured using site-directed fluorescence and ESR spectroscopy. For this purpose, we investigate the membrane-anchoring interactions of M13 coat protein in model systems consisting of phosphatidylcholine bilayers that vary in hydrophobic thickness. Mutant coat proteins are prepared with an AEDANS-labeled single cysteine residue in the hinge region of the protein or at the C-terminal side of the transmembrane helix. In addition, the fluorescence of the tryptophan residue is studied as a monitor for the N-terminal side of the transmembrane helix. The fluorescence results show that the hinge region and C-terminal side of the transmembrane helix hardly respond to hydrophobic mismatch. In contrast, the N-terminal side of the helical transmembrane domain shifts to a more apolar environment, when the hydrophobic thickness is increased. The apparent strong membrane-anchoring interactions of the C-terminus are confirmed using a mutant that contains a longer transmembrane domain. As a result of this mutation, the tryptophan residue at the N-terminal side of the helical domain clearly shifts to a more polar environment, whereas the labeled position 46 at the C-terminal side is not affected. The phenylalanines in the C-terminal part of the protein play an important role in these apparent strong anchoring interactions. This is demonstrated with a mutant in which both phenylalanines are replaced by alanine residues. The phenylalanine residues in the C-terminus affect the location in the membrane of the entire transmembrane domain of the protein.
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- 2001
25. Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer
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Tibor Páli, Derek Marsh, Marcus A. Hemminga, and Denys Bashtovyy
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Models, Molecular ,M13 bacteriophage ,Molecular model ,Lipid Bilayers ,Biophysics ,Phospholipid ,Molecular modeling ,Biochemistry ,Article ,Maleimides ,Molecular dynamics ,chemistry.chemical_compound ,Capsid ,Cysteine ,Protein Structure, Quaternary ,Spin label ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Phospholipids ,Viral coat protein ,biology ,Chemistry ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,Site-directed spin-labeling ,Site-directed spin labeling ,biology.organism_classification ,Protein Structure, Tertiary ,Crystallography ,Biofysica ,Solvation shell ,Amino Acid Substitution ,Membrane protein ,Mutation ,Capsid Proteins ,Spin Labels ,EPS ,Electron paramagnetic resonance ,Lipid-protein interaction ,Bacteriophage M13 - Abstract
The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments.
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- 2001
26. Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes
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Rudy A. Demel, Cor J. A. M. Wolfs, M. Chantal Houbiers, Marcus A. Hemminga, and Ruud B. Spruijt
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Vesicle-associated membrane protein 8 ,Blotting, Western ,Lipid Bilayers ,Biophysics ,medicine.disease_cause ,Biochemistry ,Spontaneous insertion ,Capsid ,medicine ,Escherichia coli ,Pressure ,Inner membrane ,Binding site ,Binding Sites ,biology ,Chemistry ,Vesicle ,Phosphatidylglycerols ,Cell Biology ,Proteinase K ,Fluoresceins ,Transmembrane protein ,Biofysica ,Membrane ,biology.protein ,Phosphatidylcholines ,Model membranes ,Capsid Proteins ,Binding Sites, Antibody ,EPS ,Endopeptidase K ,Bacteriophage M13 - Abstract
Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of insertion of this small protein (32 amino acids) into membranes is still unknown. Here we show that the protein is able to insert in monolayers. The limiting surface pressure of 35 mN/m for 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo. By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside. The orientation after insertion of gene 9 protein is in agreement with the ‘positive inside rule’.
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- 2001
- Full Text
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27. The effects of moisture and temperature on the ageing kinetics of pollen: interpretation based on cytoplasmic mobility
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Marcus A. Hemminga, Julia Buitink, Folkert A. Hoekstra, and Olivier Leprince
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Physiology ,Longevity ,Kinetics ,Biophysics ,Plant Science ,Activation energy ,Molecular mobility ,Spin probe ,Orders of magnitude (specific energy) ,Typha latifolia ,Botany ,Laboratorium voor Plantenfysiologie ,Water content ,Moisture ,Chemistry ,rotational motion ,Humidity ,Ageing ,Biofysica ,Pollen, rotational motion ,Chemical physics ,Pollen ,Glass ,EPS ,Laboratory of Plant Physiology ,EPR spectroscopy - Abstract
This study shows that characterization of the molecular mobility in the cytoplasm of pollen provides a new understanding of the effects of moisture and temperature on ageing rates. Using EPR spectroscopy, we determined the rotational motion of the polar spin probe, 3-carboxy-proxyl, in the cytoplasm of Typha latifolia pollen, under different temperature and moisture content conditions. Increasing the temperature resulted in faster rotational motion, analogous to faster ageing rates. With decreasing moisture content, rotational motion first decreased until a minimum was reached, after which rotational motion slightly increased again. The moisture content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm, suggesting that these parameters are causally linked. Upon melting of the intracellular glass, a twofold increase in activation energy of rotational motion and ageing rate was observed. In contrast, melting of the sucrose glass resulted in an increase in rotational motion of five orders of magnitude. The difference in rotational motion upon melting glasses of pollen or sucrose suggests that other molecules beside sugars play a role in intracellular glass formation in pollen.
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- 2000
28. Contributory presentations/posters
- Author
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N. Manoj, V. R. Srinivas, A. Surolia, M. Vijayan, K. Suguna, R. Ravishankar, R. Schwarzenbacher, K. Zeth, null Diederichs, G. M. Kostner, A. Gries, P. Laggner, R. Prassl, null Madhusudan, Pearl Akamine, Nguyen-huu Xuong, Susan S. Taylor, M. Bidva Sagar, K. Saikrishnan, S. Roy, K. Purnapatre, P. Handa, U. Varshney, B. K. Biswal, N. Sukumar, J. K. Mohana Rao, A. Johnson, Vasantha Pattabhi, S. Sri Krishna, Mira Sastri, H. S. Savithri, M. R. N. Murthy, Bindu Pillai, null Kannan, M. V. Hosur, Mukesh Kumar, Swati Patwardhan, K. K. Kannan, B. Padmanabhaa, S. Sasaki-Sugio, M. Nukaga, T. Matsuzaki, S. Karthikevan, S. Sharma, A. K. Sharma, M. Paramasivam, P. Kumar, J. A. Khan, S. Yadav, A. Srinivasan, T. P. Singh, S. Gourinath, Neelima Alam, A. Srintvasan, Vikas Chandra, Punit Kaur, Ch. Betzel, S. Ghosh, A. K. Bera, S. Bhattacharya, S. Chakraborty, A. K. Pal, B. P. Mukhopadhyay, I. Dey, U. Haldar, Asok Baneriee, Jozef Sevcik, Adriana Solovicova, K. Sekar, M. Sundaralingam, N. Genov, Dong-cai Liang, Tao Jiang, Ji-ping Zhang, Wen-rui Chang, Wolfgang Jahnke, Marcel Blommers, S. C. Panchal, R. V. Hosur, Bindu Pillay, Puniti Mathur, S. Srivatsun, Ratan Mani Joshi, N. R. Jaganathan, V. S. Chauhan, H. S. Atreya, S. C. Sahu, K. V. R. Chary, Girjesh Govil, Elisabeth Adjadj, Éric Quinjou, Nadia Izadi-Pruneyre, Yves Blouquit, Joël Mispelter, Bernadette Heyd, Guilhem Lerat, Philippe Milnard, Michel Desmadreil, Y. Lin, B. D. Nageswara Rao, Vidva Raghunathan, Mei H. Chau, Prashant Pesais, Sudha Srivastava, Evans Coutinho, Anil Saran, Leizl F. Sapico, Jayson Gesme, Herbert Lijima, Raymond Paxton, Thamarapu Srikrishnan, C. R. Grace, G. Nagenagowda, A. M. Lynn, Sudha M. Cowsik, Sarata C. Sahu, S. Chauhan, A. Bhattacharya, G. Govil, Anil Kumar, Maurizio Pellecchia, Erik R. P. Zuiderweg, Keiichi Kawano, Tomoyasu Aizawa, Naoki Fujitani, Yoichi Hayakawa, Atsushi Ohnishi, Tadayasu Ohkubo, Yasuhiro Kumaki, Kunio Hikichi, Katsutoshi Nitta, V. Rani Parvathy, R. M. Kini, Takumi Koshiba, Yoshihiro Kobashigawa, Min Yao, Makoto Demura, Astushi Nakagawa, Isao Tanaka, Kunihiro Kuwajima, Jens Linge, Seán O. Donoghue, Michael Nilges, G. Chakshusmathi, Girish S. Ratnaparkhi, P. K. Madhu, R. Varadarajan, C. Tetreau, M. Tourbez, D. Lavalette, M. Manno, P. L. San Biagio, V. Martorana, A. Emanuele, S. M. Vaiana, D. Bulone, M. B. Palma-Vittorelli, M. U. Palma, V. D. Trivedi, S. F. Cheng, W. J. Chien, S. H. Yang, S. Francis, D. K. Chang, Renn Batra, Michael A. Geeves, Dietmar J. Manstein, Joanna Trvlska, Pawel Grochowski, Maciej Geller, K. Ginalski, P. Grochowski, B. Lesyng, P. Lavalette, Y. Blouquit, D. Roccatano, A. Amadei, A. Di Nola, H. J. C. Berendsen, Bosco Ho, P. M. G. Curmi, H. Berry, D. Lairez, E. Pauthe, J. Pelta, V. Kothekar, Shakti Sahi, M. Srinivasan, Anil K. Singh, Kartha S. Madhusudnan, Fateh S. Nandel, Harpreet Kaur, Balwinder Singh, D. V. S. Jain, K. Anton Feenstra, Herman J. C. Berendsen, F. Tama, Y. -H. Sanejouand, N. Go, Deepak Sharma, Sunita Sharma, Santosh Pasha, Samir K. Brahmachari, R. Viiavaraghavan, Jyoti Makker, Sharmisllia Dey, S. Kumar, G. S. Lakshmikanth, G. Krishnamoorthy, V. M. Mazhul, E. M. Zaitseva, Borys Kierdaszuk, J. Widengren, B. Terry, Ü. Mets, R. Rigler, R. Swaminathan, S. Thamotharan, N. Yathindra, Y. Shibata, H. Chosrowjan, N. Mataga, I. Morisima, Tania Chakraharty, Ming Xiao, Roger Cooke, Paul Selvin, C. Branca, A. Faraone, S. Magazù, G. Maisano, P. Migliardo, V. Villari, Digambar V. Behere, M. Sharique Zahida Waheed Deva, M. Brunori, F. Cutruzzolà, Q. H. Gibson, C. Savino, C. Travaglini-Allocatelli, B. Vallone, Swati Prasad, Shyamalava Mazumdar, Samaresh Mitra, P. Soto, R. Fayad, I. E. Sukovataya, N. A. Tyulkova, Sh. V. Mamedov, B. Aktas, M. Canturk, B. Aksakal, R. Yilgin, K. I. Bogutska, N. S. Miroshnichenko, S. Chacko, M. DiSanto, J. A. Hypolite, Y-M. Zheng, A. J. Wein, M. Wojciechowski, T. Grycuk, J. Antosiewicz, Marc A. Ceruso, Alfredo Di Nola, Subhasis Bandvopadhvay, Bishnu P. Chatterjee, Devapriva Choudhury, Andrew Thompson, Vivian Stojanoff, Jerome Pinkner, Scott Hultgren, Stefan Khight, Delphine Flatters, Julia Goodfellow, Fumi Takazawatt, Minoru Kanehisa, Masaki Sasai, Hironori Nakamura, Wang Bao Han, Yuan Zheng, Wang Zhi Xin, Pan xin Min, Vlnod Bhakuni, Sangeeta Kulkarni, Atta Ahmad, Koodathingal Prakash, Shashi Prajapati, Alexey Surin, Tomoharu Matsumoto, Li Yang, Yuki Nakagawa, Kazumoto Kimura, Yoshiyuki Amemiya, Gennady V. Semisotnov, Hiroshi Kihara, Saad Tayyab, Salman Muzammil, Yogesh Kumar, Vinod Bhakuni, Monica Sundd, Suman Kundu, M. V. Jagannadham, Medicherla V. Jagannadham, Bina Chandani, Ruby Dhar, Lalankumar Sinha, Deepti Warrier, Sonam Mehrotra, Purnima Khandelwal, Subhendu Seth, Y. U. Sasidhar, C. Ratna Prabha, Arun Gidwani, K. P. Madhusudan, Akira R. Kinjo, Ken Nishikawa, Suvobrata Chakravarty, Raghavan Varadarajan, K. Noyelle, P. Haezebrouck, M. Joniau, H. Van Dael, Sheffali Dash, Indra Brata Jha, Rajiv Bhat, Prasanna Mohanty, A. K. Bandyopadhyay, H. M. Sonawat, Ch. Mohan Rao, Siddhartha Datta, K. Rajaraman, B. Raman, T. Ramakrishna, A. Pande, J. Pande, S. Betts, N. Asherie, O. Ogun, J. King, G. Benedek, I. V. Sokolova, G. S. Kalacheva, Masashi Sonoyama, Yasunori Yokoyama, Kunihiro Taira, Shigeki Mitaku, Chicko Nakazawal, Takanori Sasakil, Yuri Mukai, Naoki Kamo, Seema Dalal, Lynne Regan, Shigeki Mituku, Mihir Roychoudhury, Devesh Kumar, Dénes Lőrinczv, Franciska Könczöl, László Farkas, Joseph Belagyi, Christoph Schick, Christy A. Thomson, Vettai S. Ananthanarayanan, E. G. Alirzayeva, S. N. Baba-Zade, M. Michael Gromiha, M. Oobatake, H. Kono, J. An, H. Uedaira, A. Sarai, Kazufumi Takano, Yuriko Yamagata, Katsuhide Yutani, Gouri S. Jas, Victor Muñoz, James Hofrichter, William A. Eaton, Jonathan Penoyar, Philip T. Lo Verde, J. Kardos, Á. Bódi, I. Venekei, P. Závodszky, L. Gráf, András Szilágyi, Péter Závodszky, R. D. Allan, J. Walshaw, D. N. Woolfson, Jun Funahashi, Savan Gupta, M. Mangoni, P. Roccatano, Gosu Ramachandraiah, Nagasuma R. Chandra, Barbara Ciani, Derek N. Woolfson, Usha B. Nair, Kanwal J. Kaur, Dinakar M. Salunke, Chittoor P. Swaminathan, Avadhesha Surolia, A. Pramanik, P. Jonasson, G. Kratz, O. T. Jansson, P. -Å. Nygren, S. Ståhl, K. Ekberg, B. -L. Johansson, S. Uhlén, M. Uhlén, H. Jörnvall, J. Wahren, Karin Welfle, Rolf Misselwitz, Wolfgang Höhne, Heinz Welfle, L. G. Mitskevich, N. V. Fedurkina, B. I. Kurganov, Gotam K. Jarori, Haripada Maity, J. Guharay, B. Sengupta, P. K. Sengupta, K. Sridevi, S. R. Kasturi, S. P. Gupta, Gunjan Agarwal, Suzanne Kwong, Robin W. Briehl, O. I. Ismailova, N, A. Tyulkova, C. Hariharan, D. Pines, E. Pines, M. Zamai, R. Cohen-Luria, A. Yayon, A. H. Parola, M. J. Padya, G. A. Spooner, D. N. Woolfeon, Panchan Bakshi, D. K. Bharadwaj, U. Sharma, N. Srivastava, R. Barthwal, N. R. Jagannathan, Keiko Matsuda, Takaaki Nishioka, Nobuhiro Go, T. Aita, S. Urata, Y. Husimi, Mainak Majumder, Nicola G. A. Abrescia, Lucy Malinina, Juan A. Subirana, Juan Aymami, Ramón Eritxa, Miquel Coll, B. J. Premraj, R. Thenmalarchelvi, P. Satheesh Kumar, N. Gautham, Lou -Sing Kan, null Ming-Hou, Shwu-Bin Lin, Tapas Sana, Kanal B. Roy, N. Bruant, D. Flatters, R. Lavery, D. Genest, Remo Rons, Heinz Sklenar, Richard Lavery, Sudip Kundu, Dhananjay Bhattacharyya, Debashree Bandyopadhyay, Ashoke Ranjan Thakur, Rabi Majumdar, F. Barceló, J. Portugal, Sunita Ramanathan, B. J. Rao, Mahua Gliosli, N. Vinay Kumar, Umesh Varshney, Shashank S. Pataskar, R. Sarojini, S. Selvasekarapandian, P. Kolandaivel, S. Sukumar, P. Kolmdaivel, Motilal Maiti, Anjana Sen, Suman Das, Elisa Del Terra, Chiara Suraci, Silvia Diviacco, Franco Quadrifoglio, Luigi Xodo, Arghya Ray, G. Karthikeyan, Kandala V. R. Chary, Basuthkar J. Rao, Anwer Mujeeb, Thomas L. James, N. Kasyanenko, E. E. F. Haya, A. Bogdanov, A. Zanina, M. R. Bugs, M. L. Cornélio, M. Ye. Tolstorukov, Nitish K. Sanval, S. N. Tiwari, Nitish K. Sanyal, Mihir Roy Choudhury, P. K. Patel, Neel S. Bhavesh, Anna Gabrielian, Stefan Wennmalm, Lars Edman, Rudolf Rigler, B. Constantinescu, L. Radu, I. Radulcscu, D. Gazdaru, Sebastian Wärmländer, Mikael Leijon, Setsuyuki Aoki, Takao Kondo, Masahiro Ishiura, V. A. Pashinskaya, M. V. Kosevich, V. S. Shelkovsky, Yu. P. Blagoy, Ji-hua Wang, R. Malathi, K. Chandrasekhar, E. R. Kandimalla, S. Agrawal, V. K. Rastogi, M. Alcolea Palafox, Chatar Singh, A. D. Beniaminov, S. A. Bondarenko, E. M. Zdobnov, E. E. Minyat, N. B. Ulyanov, V. I. Ivanov, J. S. Singh, Kailas D. Sonawane, Henri Grosjean, Ravindra Tewari, Uddhavesh B. Sonavane, Annie Morin, Elizabeth A. Doherty, Jennifer A. Doudna, H. Tochio, S. Sato, H. Matsuo, M. Shirakawa, Y. Kyogoku, B. Javaram, Surjit B. Dixit, Piyush Shukla, Parul Kalra, Achintya Das, Kevin McConnell, David L. Beveridge, W. H. Sawyer, R. Y. S. Chan, J. F. Eccelston, Yuling Yan, B. E. Davidson, Eimer Tuite, Bengt Norden, Peter Nielsen, Masayuki Takahashi, Anirban Ghosh, Manju Bansal, Frauke Christ, Hubert Thole, Wolfgang Wende, Alfred Pingoud, Vera Pingoud, Pratibha Mehta Luthra, Ramesh Chandra, Ranjan Sen, Rodney King, Robert Weisberg, Olaf F. A. Larsen, Jos Berends, Hans A. Heus, Cornelis W. Hilbers, Ivo H. M. van Stokkum, Bas Gobets, Rienk van Grondelle, Herbert van Amerongen, HE. Sngrvan, Yu. S. Babayan, N. V. Khudaverdian, M. Gromiha, F. Pichierri, M. Aida, P. Prabakaran, K. Sayano, Saulius Serva, Eglė Merkienė, Giedrius Vilkaitis, Elmar Weinhold, Saulius Klimašauskas, Eleonora Marsich, Antonella Bandiera, Giorgio Manzini, G. Potikyan, V. Arakelyan, Yu. Babayan, Alex Ninaber, Julia M. Goodfellow, Yoichiro Ito, Shigeru Ohta, Yuzuru Husimi, J. Usukura, H. Tagami, H. Aiba, Mougli Suarez, Elia Nunes, Deborah Keszenman, E. Carmen Candreva, Per Thyberg, Zeno Földes-Papp, Amita Joshi, Dinesh Singh, M. R. Rajeswari, null Ira, M. Pregetter, H. Amenitsch, J. Chapman, B. N. Pandev, K. P. Mishra, E. E. Pohl, J. Sun, I. I. Agapov, A. G. Tonevitsky, P. Pohl, S. M. Dennison, G. P. Gorbeako, T. S. Dynbko, N. Pappavee, A. K. Mishra, Prieto Manuel, Almeida Rodrigo, Loura Luis, L. Ya. Gendel, S. Przestalski, J. Kuczera, H. Kleszczyńska, T. Kral, E. A. Chernitsky, O. A. Senkovich, V. V. Rosin, Y. M. Allakhverdieva, G. C. Papageorgiou, R. A. Gasanov, Calin Apetrei, Tudor Savopol, Marius Balea, D. Cucu, D. Mihailescu, K. V. Ramanathan, Goran Bačić, Nicolas Sajot, Norbert Garnier, Serge Crouzy, Monique Genest, Z. S. Várkonyi, O. Zsiros, T. Farkas, Z. Combos, Sophie Cribier, I. F. Fraceto, S. Schreier, A. Spisni, F. de Paula, F. Sevšek, G. Gomišček, V. Arrigler, S. Svetina, B. Žekš, Fumimasa Nomura, Miki Nagata, Kingo Takiguchi, Hirokazu Hotani, Lata Panicker, P. S. Parvathanathan, A. Ishino, A. Saitoh, H. Hotani, K. Takiguchi, S. Afonin, A. Takahashi, Y. Nakato, T. Takizawa, Dipti Marathe, Kent Jørgensen, Satinder S. Rawat, R. Rukmini, Amitabha Chattopadhyay, M. Šentiurc, J. Štrancar, Z. Stolič, K. Filipin, S. Pečar, S. C. Biswas, Satyen Sana, Anunay Samanta, Koji Kinoshita, Masahito Yamazaki, Tetsuhiko Ohba, Tai Kiuchi, null Yoshitoshi, null Kamakura, Akira Goto, Takaaki Kumeta, Kazuo Ohki, I. P. Sugar, T. E. Thompson, K. K. Thompson, R. L. Biltonen, Y. Suezaki, H. Ichinose, M. Akivama, S. Matuoka, K. Tsuchihashi, S. Gasa, P. Mattjus, J. G. Molotkovsky, H. M. Pike, R. E. Brown, Ashish Arora, Jörg H. Kleinschmidt, Lukas K. Tamm, O. G. Luneva, K. E. Kruglyakova, V. A. Fedin, O. S. Kuptsoya, J. W. Borst, N. V. Visser, A. J. W. G. Visser, T. S. Dyubko, Toshihiko Ogihara, Kiyoshi Mishima, A. L. Shvaleva, N. Č. Radenović, P. M. Minić, M. G. Jeremić, Č. N. Radenović, T. F. Aripov, E. T. Tadjibaeva, O. N. Vagina, M. V. Zamaraeva, B. A. Salakhutdinov, A. Cole, M. Poppofl, C. Naylor, R. Titball, A. K. Basak, J. T. Eaton, C. E. Naylor, N. Justin, D. S. Moss, R. W. Titball, F. Nomura, M. Nagata, S. Ishjkawa, S. Takahashi, Kaoru Obuchi, Erich Staudegger, Manfred Kriechbaum, Robert I. Lehrer, Alan J. Waring, Karl Lohner, Susanne Gangl, Bernd Mayer, Gottfried Köhler, J. Shobini, Z. Guttenberg, B. Lortz, B. Hu, E. Sackmann, N. M. Kozlova, L. M. Lukyanenko, A. N. Antonovich, E. I. Slobozhanina, Andrey V. Krylov, Yuri N. Antonenko, Elena A. Kotova, Alexander A. Yaroslavov, Subhendu Ghosh, Amal K. Bera, Sudipto Das, Eva Urbánková, Masood Jelokhani-Niaraki, Karl Freeman, Petr Jezek, P. B. Usmanov, A. Ongarbaev, A. K. Tonkikh, Peter Pohl, Sapar M. Saparov, P. Harikumar, J. P. Reeves, S. Rao, S. K. Sikdar, A. S. Ghatpande, C. Corsso, A. C. Campos de Carvalho, W. A. Varanda, C. ElHamel, E. Dé, N. Saint, G. Molle, Anurae Varshney, M. K. Mathew, E. Loots, E. Y. Isacoff, Michiki Kasai, Naohiro Yamaguchi, Paramita Ghosh, Joseph Tigyi, Gabor Tigyi, Karoly Liliom, Ricardo Miledi, Maja R. Djurisic, Pavle R. Andjus, Indira H. Shrivastava, M. S. P. Sansom, C. Barrias, P. F. Oliveira, A. C. Mauricio, A. M. Rebelo da Costa, I. A. Lopes, S. V. Fedorovich, V. S. Chubanov, M. V. Sholukh, S. V. Konev, N. Fedirko, V. Manko, M. Klevets, N. Shvinka, B. S. Prabhananda, Mamata H. Kombrabail, S. Aravamudhan, Berenice Venegas-Cotero, Ivan Ortega Blake, Zhi-hong Zhang, Xiao-jian Hu, Han-qing Zhou, Wei-ying Cheng, Hang-fang Feng, L. O. Dubitsky, L. S. Vovkanvch, I. A. Zalyvsky, E. Savio-Galimberti, P. Bonazzola, J. E. Ponce-Homos, Mario Parisi, Claudia Capurro, Roxana Toriano, Laxma G. Ready, Larry R. Jones, David D. Thomas, B. A. Tashmukhamedov, B. T. Sagdullaev, D. Heitzmann, R. Warth, M. Bleich, R. Greger, K. T. G. Ferreira, H. G. Ferreira, Orna Zagoory, Essa Alfahel, Abraham H. Parola, Zvi Priel, H. Hama-Inaba, R. Wang, K. Choi, T. Nakajima, K. Haginoya, M. Mori, H. Ohyama, O. Yukawa, I. Hayata, Nanda B. Joshi, Sridhar K. Kannurpatti, Preeti G. Joshi, Mau Sinha, Xun Shen, Tianhui Hu, Ling Bei, Menno L. W. Knetsch, Nicole Schäfers, John Sandblom, Juris Galvanovskis, Roxana Pologea-Moraru, Eugenia Kovacs, Alexandra Dinu, S. H. Sanghvi, V. Jazbinšek, G. Thiel, W. Müller, G. Wübeller, Z. Tronteli, Leš Fajmut, Marko Marhl, Milan Brumen, I. D. Volotovski, S. G. Sokolovski, M. R. Knight, Alexei N. Vasil’ev, Alexander V. Chalyi, P. Sharma, P. J. Steinbach, M. Sharma, N. D. Amin, J. Barchir, R. W. Albers, H. C. Pant, M. Balasubramanyam, M. Condrescu, J. P. Gardner, Shamci Monajembashi, Gotz Pilarczyk, K. O. Greulich, F. M. El-Refaei, M. M. Talaat, A. I. El-Awadi, F. M. Ali, Ivan Tahradník, Jana Pavelková, Alexandra Zahradniková, Boris S. Zhorov, Vettai S. Ananthanaravanan, M. Ch. Michailov, E. Neu, W. Seidenbusch, E. Gornik, D. Martin, U. Welscher, D. G. Weiss, B. R. Pattnaik, A. Jellali, V. Forster, D. Hicks, J. Sahel, H. Dreyfus, S. Picaud, Hong-Wei Wang, Sen-fang Sui, Pradeep K. Luther, John Barry, Ed Morris, John Squire, C. Sivakama Sundari, D. Balasubramanian, K. Veluraia, T. Hema Thanka Christlet, M. Xavier Suresh, V. Laretta-Garde, Dubravka Krilov, Nataša Stojanović, Janko N. Herak, Ravi Jasuja, Maria Ivanova, Rossen Mirchev, Frank A. Ferrone, David Stopar, Ruud B. Spruijt, Cor J. A. M. Wolfs, Marcus A. Hemminga, G. Arcovito, M. De Spirito, Rajendra K. Agrawal, Amy B. Heagle, Pawel Penczek, Robert Grassucci, Joachim Frank, Manjuli R. Sharma, Loice H. Jeyakumar, Sidney Fleischer, Terence Wagenknecht, Carlo Knupp, Peter M. G. Munro, Eric Ezra, John M. Squire, Koji Ichihara, Hidefumi Kitazawa, Yusuke Iguchi, Tomohiko J. Itoh, Greta Pifat, Marina Kveder, Slavko Pečar, Milan Schara, Deepak Nair, Kavita Singh, Kanury V. S. Rao, Kanwaljeet Kaur, Deepti Jain, B. Sundaravadivel, Manisha Goel, D. M. Salunke, E. I. Kovalenko, G. N. Semenkova, S. N. Cherenkevich, T. Lakshmanan, D. Sriram, S. Srinivasan, D. Loganathan, T. S. Ramalingam, J. A. Lebrón, P. J. Bjorkman, A. K. Singh, T. N. Gayatri, Ernesto R. Caffarena, J. Raul Grigera, Paulo M. Bisch, V. Kiessling, P. Fromherz, K. N. Rao, S. M. Gaikwad, M. I. Khan, C. G. Suresh, P. Kaliannan, M. Elanthiraiyan, K. Chadha, J. Payne, J. L. Ambrus, M. P. N. Nair, Madhavan P. N. Nair, S. Mahajan, K. C. Chadha, R. Hewitt, S. A. Schwartz, J. Bourguignon, M. Faure, C. Cohen-Addad, M. Neuburger, R. Ober, L. Sieker, D. Macherel, R. Douce, D. S. Gurumurthy, S. Velmurugan, Z. Lobo, Ratna S. Phadke, Prashant Desai, I. M. Guseinova, S. Yu. Suleimanov, I. S. Zulfugarov, S. N. Novruzova, J. A. Aliev, M. A. Ismayilov, T. V. Savchenko, D. R. Alieva, Petr Ilík, Roman Kouřil, Hana Bartošková, Jan Nauš, Jvoti U. Gaikwad, Sarah Thomas, P. B. Vidyasagar, G. Garab, I. Simidjiev, S. Rajagopal, Zs. Várkonyi, S. Stoylova, Z. Cseh, E. Papp, L. Mustárdy, A. Holzenburg, R. Bruder, U. K. Genick, T. T. Woo, D. P. Millar, K. Gerwert, E. D. Getzoff, Tamás Jávorfí, Győző Garab, K. Razi Naqvi, Md. Kalimullah, Jyoti Gaikwad, Manoj Semwal, Roman Kouril, Petr Ilik, Man Naus, István Pomozi, Gábor Horváth, Rüdiger Wehner, Gary D. Bernard, Ana Damjanović, Thorsten Ritz, Klaus Schulten, Wang Jushuo, Shan Jixiu, Gong Yandao, Kuang Tingyun, Zhao Nanming, Arvi Freiberg, Kõu Timpmann, Rein Ruus, Neal W. Woodbury, E. V. Nemtseva, N. S. Kudryasheva, A. G. Sizykh, V. N. Shikhov, T. V. Nesterenko, A. A. Tikhomirov, Giorgio Forti, Giovanni Finazzi, Alberto Furia, Romina Paola Barbagallo, S. Iskenderova, R. Agalarov, R. Gasanov, Miyashita Osamu, G. O. Nobuhiro, R. K. Soni, M. Ramrakhiani, Hiromasa Yagi, Kacko Tozawa, Nobuaki Sekino, Tomoyuki Iwabuchi, Masasuke Yoshida, Hideo Akutsu, A. V. Avetisyan, A. D. Kaulen, V. P. Skulachev, B. A. Feniouk, Cécile Breyton, Werner Kühlbrandt, Maria Assarsson, Astrid Gräslund, G. Horváth, B. Libisch, Z. Gombos, N. V. Budagovskaya, N. Kudryasheva, Erisa Harada, Yuki Fukuoka, Tomoaki Ohmura, Arima Fukunishi, Gota Kawai, Kimitsuna Watanabe, Jure Derganc, Bojan Božič, Saša Svetina, Boštjan Žekš, J. F. Y. Hoh, Z. B. Li, G. H. Rossmanith, E. L. de Beer, B. W. Treijtel, P. L. T. M. Frederix, T. Blangè, S. Hénon, F. Galtet, V. Laurent, E. Planus, D. Isabey, L. S. Rath, P. K. Dash, M. K. Raval, C. Ramakrishnan, R. Balaram, Milan Randic, Subhash C. Basak, Marjan Vracko, Ashesh Nandy, Dragan Amic, Drago Beslo, Sonja Nikolic, Nenad Trinajstic, J. Walahaw, Marc F. J. Lensink, Boojala V. B. Reddy, Ilya N. Shindylov, Philip E. Bourne, M. C. Donnamaria, J. de Xammar Oro, J. R. Grigera, Monica Neagu, Adrian Neagu, Matej Praprotnik, Dušanka Janežič, Pekka Mark, Lennart Nilsson, L. La Fata, Laurent E. Dardenne, Araken S. Werneck, Marçal de O. Neto, N. Kannan, S. Vishveshwara, K. Veluraja, Gregory D. Grunwald, Alexandra T. Balaban, Kanika Basak, Brian D. Gute, Denise Mills, David Opitz, Krishnan Balasubramanian, G. I. Mihalas, Diana Lungeanu, G. Macovievici, Raluca Gruia, C. Cortez-Maghelly, B. Dalcin, E. P. Passos, S. Blesic, M. Ljubisavljevic, S. Milosevic, D. J. Stratimirovic, Nandita Bachhawat, Shekhar C. Mande, A. Nandy, Ayumu Saito, Koichi Nishigaki, Mohammed Naimuddin, Takatsugu Hirokawa, Mitsuo Ono, Hirotomo Takaesu, M. I. El Gohary, Abdalla S. Ahmed, A. M. Eissa, Hiroshi Nakashima, G. P. S. Raghava, N. Kurgalvuk, O. Goryn, Bernard S. Gerstman, E. V. Gritsenko, N. N. Remmel, O. M. Maznyak, V. A. Kratasyuk, E. N. Esimbekova, D. Tchitchkan, S. Koulchitsky, A. Tikhonov, A. German, Y. Pesotskaya, S. Pashkevich, S. Pletnev, V. Kulchitsky, Umamaheswar Duvvuri, Sridhar Charagundla, Rahim Rizi, John S. Leigh, Ravinder Reddy, Mahesh Kumar, O. Coshic, P. K. Julka, O. K. Rath, NR. Jagannathan, Karina Roxana Iliescu, Maria Sajin, Nicolcta Moisoi, Ileana Petcu, A. I. Kuzmenko, R. P. Morozova, I. A. Nikolenko, G. V. Donchenko, M. K. Rahman, M. M. Ahmed, Takehiro Watanabe, Y. Rubin, H. Gilboa, R. Sharony, R. Ammar, G. Uretzky, M. Khubchandani, H. N. Mallick, V. Mohan Kumar, Arijitt Borthakur, Erik M. Shapiro, M. Gulnaz Begum, Mahaveer N. Degaonkar, S. Govindasamy, Ivan Dimitrov, T. A. Kumosani, W. Bild, I. Stefanescu, G. Titescu, R. Iliescu, C. Lupusoru, V. Nastasa, I. Haulica, Gopal Khetawat, N. Faraday, M. Nealen, S. Noga, P. F. Bray, T. V. Ananieva, E. A. Lycholat, MV. Kosevich, S. G. Stepanyan, S. V. Antonyuk, R. Khachatryan, H. Arakelian, A. Kumar, S. Ayrapetyan, V. Mkheyan, S. Agadjanyan, A. Khachatryan, S. S. Rajan, V. Kabaleeswaran, Geetha Gopalakrishnan, T. R. Govindachari, Meera Ramrakhiani, Phillip Lowe, Andrew Badley, David C. Cullen, H. Hermel, W. Schmahl, H. Möhwald, Nirmalya Majumdar, Joydip Das, András Dér, Loránd Kelemen, László Oroszi, András Hámori, Jeremy J. Ramsden, Pál Ormos, D. Savitri, Chanchal K. Mitra, Toshio Yanagida, Seiji Esaki, Yuji Kimura, Tomoyuki Nishida, Yosiyuki Sowa, M. Radu, V. K. Koltover, Ya. I. Estrin, L. A. Kasumova, V. P. Bubnov, E. E. Laukhina, Rajiv Dotta, M. Degaonkar, P. Raghunathan, Rama Jayasundar, Pavel Novák, Milan Marko, Ivan Zahradník, Hiroaki Hirata, Hidetake Miyata, J. Balaji, P. Sengupta, S. Maiti, M. Gonsalves, A. L. Barker, J. V. Macpherson, D. O’Hare, C. P. Winlove, P. R. Unwin, R. Phillip, S. Banerjee, G. Ravindra Kumar, K. Nagayaka, R. Danev, S. Sugitani, K. Murata, Michael Gősch, H. Blom, P. Thyberg, Z. Földes-Papp, G. Björk, J. Holm, T. Heino, Masashi Yokochi, Fuyuhiko Inagaki, Masami Kusunoki, E. K. Matthews, J. Pines, Yu. P. Chukova, Vitaly K. Koltover, Geetanjali Bansal, Uma Singh, M. P. Bansal, Kotoko Nakata, Tastuya Nakano, Tsuguchika Kaminuma, B. P. S. Kang, U. Singh, Bonn Kirn, Neja Potocnik, Vito Stare, Latal Shukla, V. Natarajan, T. P. A. Devasagayam, M. D. Sastry, P. C. Kesavan, R. Sayfutdinov, V. V. Adamovich, D. Yu. Rogozin, A. G. Degermendzhy, C. L. Khetrapal, G. A. Nagana Gowda, Kedar Nath Ghimire, Ishida Masaru, H. Fujita, S. Ishiwata, Y. Kishimoto, S. Kawahara, M. Suzuki, H. Mori, M. Mishina, Y. Kirino, H. Ohshima, A. S. Dukhin, V. N. Shilov, P. J. Goetz, and R. K. Mishra
- Subjects
0303 health sciences ,biology ,General Medicine ,010402 general chemistry ,01 natural sciences ,Horseradish peroxidase ,General Biochemistry, Genetics and Molecular Biology ,0104 chemical sciences ,03 medical and health sciences ,Biochemistry ,Manganese porphyrin ,biology.protein ,Enzyme reconstitution ,General Agricultural and Biological Sciences ,030304 developmental biology - Published
- 1999
29. Imbibitional leakage from anhydrobiotes revisited
- Author
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Folkert A. Hoekstra, A.C. van Aelst, Marcus A. Hemminga, and Elena A. Golovina
- Subjects
Laboratorium voor Plantencelbiologie ,EPR spin probe technique ,Physiology ,Imbibition ,Typha latifolia L ,Injury ,Plant Science ,Spin probe ,Phase (matter) ,Botany ,Electron microscopy ,Laboratorium voor Plantenfysiologie ,Leakage (electronics) ,Phase transition ,Membranes ,Moisture ,Chemistry ,Humidity ,Laboratory of Plant Cell Biology ,Membrane ,Germination ,Biophysics ,Anhydrobiotes ,Pollen ,EPS ,Leakage ,Laboratory of Plant Physiology - Abstract
Dry desiccation-tolerant organ(ism)s leak cellular solutes when placed in water. Elevated temperatures at imbibition and elevated initial moisture contents reduce the leakage and promote growth. We have re-examined the effects of imbibitional stress imposed on cattail (Typha latifolia L.) pollen as a model anhydrobiotic system. A nitroxide spin probe technique and electron microscopy were used, allowing study of the permeability of the plasma membrane together with its visual intactness. Imbibitional leakage can be transient, or prolonged when associated with membrane damage. During the first 15 s of rehydration in medium, plasma membranes of pre-humidified pollen were highly permeable but became less permeable thereafter. The resulting transient leakage may affect vigour as measured by the rate of fresh weight increase, but did not reduce germination. A permanent, high permeability was observed when dry pollen was plunged into medium at low temperatures. This led to cell death and is associated with a phase change of the membranes from gel to liquid crystalline during imbibition. Freeze-fracture images indicate that the damage to plasma membranes is mechanically imposed by the pressure of the penetrating water rather than occurring structurally by a phase separation of membrane components. We suggest that a high rigidity of the plasma membranes in the gel phase at imbibition underlies imbibitional damage.
- Published
- 1999
30. Mobility in Maltose−Water Glasses Studied with 1H NMR
- Author
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Marcus A. Hemminga, I.J. van den Dries, and D. van Dusschoten
- Subjects
chemistry.chemical_compound ,chemistry ,Materials Chemistry ,Analytical chemistry ,Proton NMR ,Molecule ,Maltose ,Physical and Theoretical Chemistry ,Glass transition ,Water content ,Surfaces, Coatings and Films ,Relaxation behavior - Abstract
We have studied the molecular mobility of the water and carbohydrate protons in maltose samples as a function of water content and temperature using 1H NMR. In the NMR signal, slow decaying and fast decaying fractions of protons are distinguished as arising from mobile and immobile (τc > 3 μs) protons, respectively. The assignment of these fractions in terms of water and maltose protons is temperature dependent. By analyzing the relaxation behavior of the mobile protons, the mobility of the water molecules is determined. The mobility of water molecules increases with water content and temperature, and at the glass transition, a small break in mobility is observed, indicating that the water molecules slightly sense the glass transition. The method of second moments gives information about the mobility of the immobile protons. Upon cooling, the glass transition is marked by a decrease in the temperature dependence of the mobility of the hydroxyl protons of maltose. This suggests that a stable hydrogen-bond ...
- Published
- 1998
31. Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen
- Author
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Marcus A. Hemminga, Mireille Maria Anna Elisabeth Claessens, Folkert A. Hoekstra, and Julia Buitink
- Subjects
Nitroxide mediated radical polymerization ,Physiology ,Chemistry ,Analytical chemistry ,Mineralogy ,Plant Science ,law.invention ,Spin probe ,Viscosity ,Differential scanning calorimetry ,law ,Genetics ,Electron paramagnetic resonance ,Water content ,Rotational correlation time ,Intracellular - Abstract
Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativumL.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10−3 s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2Azz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2Azzmeasurements can provide an estimate of the optimum storage conditions.
- Published
- 1998
32. Mobility of Lipids in Low Moisture Bread as Studied by NMR
- Author
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G. Roudaut, H. van As, M. Le Meste, D. van Dusschoten, and Marcus A. Hemminga
- Subjects
Mobility ,Moisture ,Chemistry ,Diffusion ,Relaxation (NMR) ,Biophysics ,Mineralogy ,Bread ,Atmospheric temperature range ,Lipids ,Biochemistry ,Viscosity ,Biofysica ,Chemical engineering ,Phase (matter) ,Volume fraction ,Glass ,Water content ,Food Science - Abstract
The mobility of the lipids contained in glassy bread was studied with low resolution 1 H-NMR tomeasure their relaxation times (T 1 and T 2 ) and their translational diVusion coeYcient (D), as afunction of temperature and water content. The mobility of lipids detected with this method isindependent of the water content of the samples. The behaviour of lipids in bread is observed to becomparable to that of lipids in bulk fat in the same temperature range. D measured for lipids ismuch higher than the values for water soluble solutes in glasses as provided by the literature. It wasconcluded that the lipids were distributed in globules which are dispersed in the glassy bread matrixand within which they diVuse. O 1998 Academic Press Keywords : mobility, glass, diVusion, lipids, bread. INTRODUCTION on the diVerence between the respective properties(modulus, viscosity) of the continuous and dis-Most food products are rather complex systems persed phases. The overall stability of a foodwhich can be either homogeneous or composed of product is known to be controlled by the physicaldiVerent phases, depending on their composition. state of its constituent phases. From a chemicalThe contribution of ingredients, such as lipids, to point of view, when the dispersed phase is en-the material properties is dependent upon their trapped within a glassy matrix, it may be protectedrepartition i.e. either homogeneously distributed from the outside environment. Indeed, stability isand eventually interacting with the other in- often associated with the glassy and crystallinegredients, or organised in a dispersed phase. While states. The glassy state is a non-equilibrium statein a dispersed phase, the contribution depends on stabilised by the reduced molecular mobility; how-the volume fraction
- Published
- 1998
33. Mimicking initial interactions of bacteriophage M13 coat protein disassembly in model membrane systems
- Author
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Marcus A. Hemminga, Ruud B. Spruijt, Cor J. A. M. Wolfs, and David Stopar
- Subjects
Circular dichroism ,Lipid Bilayers ,Biophysics ,Biochemistry ,Bacteriophage ,chemistry.chemical_compound ,Protein structure ,Capsid ,Naphthalenesulfonates ,Life Science ,Sodium dodecyl sulfate ,Lipid bilayer ,biology ,Chemistry ,Vesicle ,Circular Dichroism ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,Site-directed spin labeling ,biology.organism_classification ,Membrane ,Spectrometry, Fluorescence ,Biofysica ,Models, Chemical ,Phosphatidylcholines ,Capsid Proteins ,EPS ,Spin Trapping ,Bacteriophage M13 - Abstract
The structure and changes in environment of the M13 major coat protein were studied in model systems, mimicking the initial molecular process of the phage disassembly. For this purpose we have systematically studied protein associations with various detergents and lipids in two different coat protein assemblies: phage particles and S-forms. It is remarkable that the major coat protein can change its conformation to accommodate three distinctly different environments: phage filament, S-form, and membrane-bound form. The structural and environmental changes during this protein transformations were studied by site-directed spin labeling, fluorescence labeling, and CD spectroscopy in different membrane model systems. The phage particles were disrupted only by strong ionic detergents [sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide and (CTAB)] but were not affected by sodium cholate and sodium deoxycholate, nonionic detergents, and dilauroyl-l-alpha-phosphatidylcholine (DLPC) lipid bilayers. Conversion of the phage particles into S-forms by addition of chloroform rendered the coat protein accessible for the association with different ionic and nonionic detergents, as well as DLPC lipids. The disruption of the S-form by all detergents studied was instantaneous but was slower with DLPC vesicles. Only small unilamellar vesicles effectively solubilized the S-form. The data suggest that the viral protein coat is inherently unstable when the major coat protein is exposed to amphiphilic molecules. During conversion from the phage to the S-form, and subsequently to the membrane-bound form, the coat protein undergoes pronounced changes in environment, and in response the alpha-helix content decreases and the local protein structure changes dramatically. This adaptation of the protein conformation enables a stable association of the protein with the membrane.
- Published
- 1998
34. Sensitivity of saturation transfer electron spin resonance extended to extremely slow mobility in glassy materials
- Author
-
Ivon J. van den Dries, Marcus A. Hemminga, and P Adrie De Jager
- Subjects
Glycerol ,Nuclear and High Energy Physics ,Physics and Physical Chemistry of Foods ,Chemistry ,TEMPOL ,Analytical chemistry ,Biophysics ,Electron ,Condensed Matter Physics ,Biochemistry ,Molecular physics ,Spectral line ,law.invention ,Viscosity ,Glucose ,Biofysica ,law ,Electron paramagnetic resonance ,Spin label ,Glass transition ,Correlation time ,Rotational correlation time ,Spin-½ - Abstract
A novel extension of the saturation transfer (ST) ESR technique that enables the determination of extremely long rotational correlation times of nitroxide spin labels up to values around 10(4) s is proposed. The method is based on the observation that the integral of ST-ESR spectra is sensitive to the spin-lattice relaxation time of the electron of the spin label, which in turn is directly dependent upon the rotational correlation time. The method is applied to the spin label TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) in glycerol. From the known viscosity data and the related rotational correlation times of the TEMPOL spin label in glycerol, the rotational correlation times of unknown samples can be determined. The method is especially applicable to systems with a very high viscosity, such as glassy materials. The method is applied to a 20 wt% glucose-water mixture in the glassy state, giving a value for the highest limiting rotational correlation time of about 10(3) s at a temperature of 45 K below the glass transition temperature of this system. This is an extension by six decades for the rotational correlation time, as compared to the current application of ST-ESR. Copyright 1998 Academic Press.
- Published
- 1998
35. Editorial: NMR in soil science
- Author
-
Marcus A. Hemminga and Peter Buurman
- Subjects
WIMEK ,Biofysica ,Materials science ,Laboratorium voor Bodemkunde en geologie ,Biophysics ,Life Science ,Soil Science ,Soil science ,Laboratory of Soil Science and Geology - Published
- 1997
36. Membrane Location of Spin-Labeled M13 Major Coat Protein Mutants Determined by Paramagnetic Relaxation Agents
- Author
-
Tibor Páli, Marcus A. Hemminga, David Stopar, Derek Marsh, and Kitty A J Jansen
- Subjects
Phospholipid ,Biochemistry ,Protein Structure, Secondary ,law.invention ,chemistry.chemical_compound ,Capsid ,Protein structure ,Nickel ,law ,Cysteine ,Spin label ,Electron paramagnetic resonance ,Binding Sites ,Chemistry ,Bilayer ,Cell Membrane ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,Transmembrane protein ,Oxygen ,Crystallography ,Transmembrane domain ,Membrane ,Liposomes ,Mutagenesis, Site-Directed ,Capsid Proteins ,Spin Labels - Abstract
Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. These were used for site-directed spin-labeling to determine the location and assembly of the major coat protein incorporated in bilayer membranes of dioleoylphosphatidylcholine. The membrane location of the spin-labeled cysteine residues was studied with molecular oxygen and Ni2+ ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-saturation electron spin resonance (ESR) spectroscopy. The section of the protein around Thr36 is situated at the center of the membrane. Residue Thr46 is placed at the membrane surface in the phospholipid head group region with a short C-terminal section, including Ala49, extending into the aqueous phase. Residue Ala25 is then positioned consistently in the head group region of the apposing lipid monolayer leaflet. These positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydrophobic section (residues 25-46), and then drop abruptly in the aqueous phase (residue 49). Additionally, the strong immobilization and low oxygen accessibility of residue 25 are attributed to steric restriction at the hinge region between the transmembrane and N-terminal amphipathic helices. Sequence-specific modulations of the ESR parameters are also observed. Relatively low oxygen accessibilities in the hydrophobic region suggest intermolecular associations of the transmembrane helices, in agreement with saturation transfer ESR studies of the overall protein mobility. Relaxation enhancements additionally reveal a Ni2+ binding site in the N-terminal domain that is consistent with a surface orientation of the amphipathic helix.
- Published
- 1997
37. Conventional and saturation-transfer EPR of spin-labeled mutant bacteriophage M13 coat protein in phospholipid bilayers
- Author
-
Ruud N.H. Konings, Marcus A. Hemminga, Wim F. Wolkers, Ruud B. Spruijt, and Anita Kaan
- Subjects
Models, Molecular ,Protein Conformation ,Lipid Bilayers ,Biophysics ,Protein dynamics ,Biochemistry ,law.invention ,Capsid ,Nuclear magnetic resonance ,Protein structure ,law ,Electron paramagnetic resonance ,Spin label ,Lipid bilayer ,Phospholipids ,Chemistry ,Bilayer ,Electron Spin Resonance Spectroscopy ,Rotation around a fixed axis ,Membrane Proteins ,Cell Biology ,Site-directed spin labeling ,M13 coat protein ,(ST)-EPR ,Crystallography ,Biofysica ,Protein conformation ,Membrane protein ,Mutation ,Capsid Proteins ,Spin Labels ,EPS - Abstract
A mutant of bacteriophage M13 was prepared in which a cysteine residue was introduced at position 25 of the major coat protein. The mutant coat protein was spin-labeled with a nitroxide derivative of maleimide and incorporated at different lipid-to-protein (L/P) ratios in DOPC or DOPG. The rotational dynamics of the reconstituted mutant coat protein was studied using EPR and saturation transfer (ST) EPR techniques. The spectra are indicative for an anisotropic motion of the maleimide spin label with a high order parameter (S=0.94). This is interpreted as a wobbling motion of the spin label with a correlation time of about 10−6 to 10−5 s within a cone, and a rotation of the spin label about its long molecular axis with a correlation time of about 10−7 s. The wobbling motion is found to correspond generally to the overall rotational motion of a coat protein monomer about the normal to the bilayer. This motion is found to be sensitive to the temperature and L/P ratio. The high value of the order parameter implies that the spin label experiences a strong squeezing effect by its local environment, that reduces the amplitude of the wobbling motion. This squeezing effect is suggested to arise from a turn structure in the coat protein from Gly23 to Glu20.
- Published
- 1997
38. Local Dynamics of the M13 Major Coat Protein in Different Membrane-Mimicking Systems
- Author
-
Marcus A. Hemminga, Cor J. A. M. Wolfs, Ruud B. Spruijt, and David Stopar
- Subjects
Protein Conformation ,Vesicle ,Bilayer ,Electron Spin Resonance Spectroscopy ,Membrane Proteins ,Sodium Dodecyl Sulfate ,Cholic Acids ,Cholic Acid ,Site-directed spin labeling ,Biology ,Biochemistry ,Micelle ,Transmembrane protein ,Capsid ,Mutagenesis, Site-Directed ,Phosphatidylcholines ,Capsid Proteins ,Cysteine ,Sodium Cholate ,Lipid bilayer ,Protein secondary structure ,Micelles - Abstract
The local environment of the transmembrane and C-terminal domain of M13 major coat protein was probed by site-directed ESR spin labeling when the protein was introduced into three membrane-mimicking systems, DOPC vesicles, sodium cholate micelles, and SDS micelles. For this purpose, we have inserted unique cysteine residues at specific positions in the transmembrane and C-terminal region, using site-directed mutagenesis. Seven viable mutants with reasonable yield were harvested: A25C, V31C, T36C, G38C, T46C, A49C, and S50C. The mutant coat proteins were indistinguishable from wild type M13 coat protein with respect to their conformational and aggregational properties. The ESR data suggest that the amino acid positions 25 and 46 of the coat protein in DOPC vesicles are located close to the membrane-water interface. In this way the lysines at positions 40, 43, and 44 and the phenylalanines at positions 42 and 45 act as hydrophilic and hydrophobic anchors, respectively. The ESR spectra of site specific maleimido spin-labeled mutant coat proteins reconstituted into DOPC vesicles and solubilized in sodium cholate or SDS indicate that the local dynamics of the major coat protein is significantly affected by its structural environment (micellar vs bilayer), location (aqueous vs hydrophobic), and lipid/protein ratio. The detergents SDS and sodium cholate sufficiently well solubilize the major coat protein and largely retain its secondary structure elements. However, the results indicate that they have a poorly defined protein-amphiphilic structure and lipid-water interface as compared to bilayers and thus are not a good substitute for lipid bilayers in biophysical studies.
- Published
- 1996
39. Multiple pathway relaxation enhancement in the system composed of three paramagnetic nitroxide radical-Ln3+-O2
- Author
-
Heidrun Jäger, Marcus A. Hemminga, Petra Lueders, Gunnar Jeschke, and Maxim Yulikov
- Subjects
Nitroxide mediated radical polymerization ,lanthanide ,model membranes ,Biophysics ,spin labels ,lipid-bilayers ,law.invention ,Ion ,Paramagnetism ,Nuclear magnetic resonance ,metmyoglobin variants ,law ,General Materials Science ,Physical and Theoretical Chemistry ,Lipid bilayer ,Electron paramagnetic resonance ,interspin distances ,Spins ,Chemistry ,alpha-helical peptides ,Relaxation (NMR) ,Site-directed spin labeling ,hydrophobic mismatch ,saturation recovery epr ,Crystallography ,Biofysica ,oxygen - Abstract
Longitudinal relaxation of nitroxide spin-labels has been measured for a membrane-incorporated alpha-helical polypeptide in the presence and absence of residual amounts of membrane-dissolved O-2 and paramagnetic Dy3+ ions. Such a model system, containing three different types of paramagnetic species, provides an important example of nonadditivity of two different relaxation channels for the nitroxide spins.
- Published
- 2012
40. Secondary structure of M13 coat protein in phospholipids studied by circular dichroism, Raman, and Fourier transform infrared spectroscopy
- Author
-
Marcus A. Hemminga, Cees Otto, Johan C. Sanders, Parvez I. Haris, and Dennis Chapman
- Subjects
Circular dichroism ,Chemistry ,Circular Dichroism ,Biophysics ,Synthetic membrane ,Membrane Proteins ,Infrared spectroscopy ,Spectrum Analysis, Raman ,Biochemistry ,Protein Structure, Secondary ,Crystallography ,Capsid ,Biofysica ,Membrane ,Protein structure ,Spectroscopy, Fourier Transform Infrared ,Life Science ,Fourier transform infrared spectroscopy ,Lipid bilayer ,Protein secondary structure ,METIS-129431 ,Bacteriophage M13 - Abstract
There is considerable uncertainty about the precise secondary structure adopted by the M13 coat protein when embedded in a phospholipid bilayer. Circular dichroism (CD) spectroscopy suggests that a major change in the structure of the coat protein occurs upon membrane insertion. It is reported that the structure of the protein in the membrane has only about 50% alpha-helix, the rest being mainly in a beta-sheet conformation, whereas the protein is almost completely alpha-helical when intact in the phage. In this study we have undertaken a spectroscopic analysis using Fourier transform infrared, Raman, and CD spectroscopy to characterize the secondary structure of M13 coat protein when present in membranes consisting of dioleoylphosphatidylglycerol and dimyristoylphosphatidylglycerol. In sharp contrast to earlier CD studies, our results indicate that the coat protein in its membrane-embedded state has a very high alpha-helical content with virtually no beta-sheet structures present. This result indicates that the structures of the coat protein when intact in the phage or when embedded in the membrane are similar. Although our results differ from earlier CD studies, they are consistent with a recent NMR study, which showed that the M13 coat protein in sodium dodecyl sulfate micelles is primarily alpha-helical with no evidence for beta-sheet structure [Henry, G. D., & Sykes, B.D. (1992) Biochemistry 31, 5284-5297]. These results lead to the conclusion that the M13 coat protein can insert from the membrane-bound state into a virus particle with a similar secondary structure, without large energy implications.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
41. Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins
- Author
-
Marieke Schor, D. Peter Tieleman, A. Baumgaertner, Marcus A. Hemminga, and Werner L. Vos
- Subjects
Models, Molecular ,conformation ,spectroscopy ,Time Factors ,Protein Conformation ,Lipid Bilayers ,fret ,Membrane biology ,Biophysics ,Energy transfer (FRET) ,Molecular Dynamics Simulation ,01 natural sciences ,orientation ,03 medical and health sciences ,Molecular dynamics ,Protein structure ,energy-transfer ,Naphthalenesulfonates ,0103 physical sciences ,Fluorescence Resonance Energy Transfer ,Lipid bilayer ,major coat protein ,transmembrane alpha-helix ,030304 developmental biology ,Fluorescent Dyes ,Probability ,0303 health sciences ,Original Paper ,model ,010304 chemical physics ,Chemistry ,EPS-2 ,Tryptophan ,association ,Membrane Proteins ,General Medicine ,Computer simulation ,Fluorescence ,Förster resonance energy transfer ,Biofysica ,Membrane protein ,Biochemistry ,Models, Chemical ,Side-chain conformations ,Mutation ,Phosphatidylcholines ,Capsid Proteins ,bilayers - Abstract
Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, alpha-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previous FRET experiments. The results indicate that AEDANS is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein.
- Published
- 2010
42. Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths
- Author
-
Bart van Oort, Ivo H. M. van Stokkum, Sergey P. Laptenok, Arie van Hoek, Rob B. M. Koehorst, Herbert van Amerongen, Marcus A. Hemminga, Ruud B. Spruijt, Biophysics Photosynthesis/Energy, and LaserLaB - Energy
- Subjects
Models, Molecular ,Time Factors ,Light ,Lipid Bilayers ,Analytical chemistry ,Membrane-bound water ,charge-transfer fluorescence ,hydration dynamics ,Cell membrane ,chemistry.chemical_compound ,2-Naphthylamine ,Membrane-embedded M13 coat protein ,Image Processing, Computer-Assisted ,Lipid bilayer ,Bilayer ,EPS-3 ,General Medicine ,Fluorescence ,Membrane ,medicine.anatomical_structure ,Biofysica ,Time-resolved spectroscopy ,solvation ,Laurdan ,SDG 6 - Clean Water and Sanitation ,m13 ,Dynamic stokes shift ,Phospholipid ,Biophysics ,laurdan ,relaxation ,excited-state ,medicine ,major coat protein ,n-terminal domain ,Membrane polarity ,Fluorescent Dyes ,Original Paper ,Staining and Labeling ,Cell Membrane ,Streak camera picosecond fluorescence ,Membrane Proteins ,Hydrogen Bonding ,Spectrometry, Fluorescence ,chemistry ,Solvent relaxation ,Solvents ,prodan - Abstract
Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein-lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane-water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains. © 2009 The Author(s).
- Published
- 2010
43. Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure
- Author
-
Johan C. Sanders, Derek Marsh, Marcus A. Hemminga, and Sjaak J. C. J. Peelen
- Subjects
Phosphatidylglycerol ,Fourier Analysis ,Spectrophotometry, Infrared ,Protein Conformation ,Lipid Bilayers ,Electron Spin Resonance Spectroscopy ,Synthetic membrane ,Phospholipid ,Phenol extraction ,Biochemistry ,law.invention ,Crystallography ,chemistry.chemical_compound ,Capsid ,chemistry ,law ,Phosphatidylcholines ,Bacteriophages ,lipids (amino acids, peptides, and proteins) ,Dimyristoylphosphatidylcholine ,Electron paramagnetic resonance ,Spin label ,Protein secondary structure ,Stoichiometry - Abstract
Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horvath, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1992
44. Introduction to NMR
- Author
-
Marcus A. Hemminga
- Subjects
Glossary ,Chemical engineering ,Computer science ,Management science ,Food Science ,Biotechnology - Abstract
This article provides an introduction to the basic theory and standard experimental techniques of nuclear magnetic resonance spectroscopy, to aid understanding of the following papers outlining specific applications of these techniques in various aspects of food science research, development and product quality control. A complementary Glossary of terms can be found at the end of the issue.
- Published
- 1992
45. Spectroscopy of lipid-protein interactions: structural aspects of two different forms of the coat protein of bacteriophage M13 incorporated in model membranes
- Author
-
Johan C. Sanders, Ruud B. Spruijt, and Marcus A. Hemminga
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,biology ,Chemistry ,Spectrum Analysis ,Phospholipid ,Biophysics ,Cell Biology ,Coat protein ,biology.organism_classification ,Biochemistry ,Protein–protein interaction ,Bacteriophage ,chemistry.chemical_compound ,Membrane ,Biofysica ,Life Science ,Computer Simulation ,Spectral analysis ,Fourier transform infrared spectroscopy ,Spectroscopy ,Phospholipids ,Bacterial Outer Membrane Proteins ,Bacteriophage M13 - Published
- 1992
46. SDSL-ESR-based protein structure characterization
- Author
-
Janez Štrancar, Aleh Kavalenka, Ajasja Ljubetič, Marcus A. Hemminga, and Iztok Urbančič
- Subjects
Models, Molecular ,Protein Conformation ,Spin labelling ,Biophysics ,Computational biology ,small-angle scattering ,molecular-dynamics simulations ,Protein structure ,intrinsically unstructured proteins ,Pancreatic lipase ,Humans ,ТЕХНИЧЕСКИЕ И ПРИКЛАДНЫЕ НАУКИ. ОТРАСЛИ ЭКОНОМИКИ::Электроника. Радиотехника [ЭБ БГУ] ,Binding Sites ,biology ,Chemistry ,EPS-2 ,Electron Spin Resonance Spectroscopy ,crystal-structure ,Proteins ,General Medicine ,computer.file_format ,Site-directed spin labeling ,Protein Data Bank ,side-chain conformation ,Characterization (materials science) ,Natively Unfolded Proteins ,natively unfolded proteins ,Biofysica ,Biochemistry ,pancreatic lipase ,membrane-proteins ,biology.protein ,epr spectroscopy ,Spin Labels ,computer ,biosystem complexity - Abstract
As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.
- Published
- 2009
47. Viruses: incredible nanomachines. New advances with filamentous phages
- Author
-
Marcus A. Hemminga, Werner L. Vos, Rob B. M. Koehorst, Cor J. A. M. Wolfs, Petr V. Nazarov, David Stopar, and Ruud B. Spruijt
- Subjects
Inovirus ,M13 bacteriophage ,viruses ,domain ,Molecular Sequence Data ,Membrane biology ,Biophysics ,membrane-protein ,Review ,Membrane protein anchoring ,ff fd ,Bacteriophage ,Cell membrane ,Viral Proteins ,nmr-spectroscopy ,medicine ,site ,Nanobiotechnology ,Nanotechnology ,Amino Acid Sequence ,Lipid bilayer ,major coat protein ,transmembrane alpha-helix ,Major coat protein ,Site-directed labelling ,biology ,Staining and Labeling ,EPS-2 ,Cell Membrane ,General Medicine ,dynamics ,biology.organism_classification ,display ,medicine.anatomical_structure ,Biofysica ,Biochemistry ,Membrane protein ,Bionanotechnology ,bacteriophage m13 ,Biotechnology - Abstract
During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account.
- Published
- 2009
48. Membrane protein frustration: protein incorporation into hydrophobic mismatched binary lipid mixtures
- Author
-
David Stopar, Ruud B. Spruijt, and Marcus A. Hemminga
- Subjects
Circular dichroism ,spectroscopy ,Protein domain ,Lipid Bilayers ,domain ,Biophysics ,law.invention ,Hydrophobic mismatch ,law ,Electron paramagnetic resonance ,Lipid bilayer ,major coat protein ,fluid ,M13 bacteriophage ,biology ,Chemistry ,EPS-2 ,phase-transitions ,Membrane ,Electron Spin Resonance Spectroscopy ,Temperature ,biology.organism_classification ,phosphatidylcholines ,Protein Structure, Tertiary ,Crystallography ,acyl-chain ,Biofysica ,Membrane protein ,Mutation ,Capsid Proteins ,bacteriophage m13 ,Hydrophobic and Hydrophilic Interactions ,solubilization ,bilayers - Abstract
Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.
- Published
- 2009
49. Structural properties of a peptide derived from H+-V-ATPase subunit a
- Author
-
Valérie Réat, Alain Milon, Louic S. Vermeer, Marcus A. Hemminga, Institut de Chimie de Strasbourg, Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées
- Subjects
Models, Molecular ,Circular dichroism ,Peptide conformation ,domain ,01 natural sciences ,Biochemistry ,Protein Structure, Secondary ,Protein structure ,Proton transport ,Histidine titration ,Protein secondary structure ,Peptide sequence ,ComputingMilieux_MISCELLANEOUS ,proton translocation channel ,0303 health sciences ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Chemistry ,EPS-2 ,magnetic-resonance ,vacuolar (h+)-atpases ,Hydrogen-Ion Concentration ,Peptide Conformation ,Transmembrane domain ,mediated cross-linking ,Biofysica ,Vacuolar Proton-Translocating ATPases ,spectroscopy ,Saccharomyces cerevisiae Proteins ,topology ,Stereochemistry ,Molecular Sequence Data ,Biophysics ,Saccharomyces cerevisiae ,surfaces ,010402 general chemistry ,Biophysical Phenomena ,03 medical and health sciences ,Histidine ,Amino Acid Sequence ,transmembrane segments ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,V-ATPase subunit a ,Cell Biology ,Peptide Fragments ,NMR ,0104 chemical sciences ,Protein Subunits ,Crystallography ,nmr-spectra - Abstract
The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H+-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously suggested based on NMR studies of a similar peptide in DMSO. The pKa of both histidine residues that are important for proton transport was measured in water and in SDS. The differences that are found demonstrate that the histidine residues interact with the SDS polar heads. In detergent, circular dichroism data indicate that the secondary structure of the peptide depends on the pH and the type of detergent used. Using solid-state NMR, it is shown that the peptide is immobile in phospholipid bilayers, which means that it is probably not a single transmembrane helix in these samples. The environment is important for the structure of TM7, so in subunit a it is probably held in place by the other transmembrane helices of this subunit.
- Published
- 2009
50. The in situ aggregational and conformational state of the major coat protein of bacteriophage M13 in phospholipid bilayers mimicking the inner membrane of host Escherichia coli
- Author
-
Marcus A. Hemminga and Ruud B. Spruijt
- Subjects
Circular dichroism ,Conformational change ,Protein Conformation ,Lipid Bilayers ,Molecular Sequence Data ,Phospholipid ,Biology ,medicine.disease_cause ,Coliphages ,Biochemistry ,Bacteriophage ,chemistry.chemical_compound ,Capsid ,Escherichia coli ,medicine ,Inner membrane ,Amino Acid Sequence ,Phospholipids ,Bilayer ,Cell Membrane ,Membrane Proteins ,Membranes, Artificial ,biology.organism_classification ,Spectrometry, Fluorescence ,Monomer ,chemistry ,Capsid Proteins - Abstract
The major coat protein of bacteriophage M13 has been reconstituted into phospholipids with a composition comparable to that found in the host (Escherichia coli) inner membrane. Reconstitution experiments have revealed conditions in which the alpha-oligomeric state is favored over the beta-polymeric state. Discrimination between the two states of the membrane-bound coat protein (alpha-oligomeric and beta-polymeric states) has been achieved using high-performance size-exclusion chromatography and circular dichroism. Interprotein electrostatic interactions, probably induced by head-tail binding, are initiated and facilitating the aggregation-related conformational change process, in which alpha-oligomeric coat protein is converted into beta-polymeric coat protein. A model for this beta-polymerization process of the coat protein is presented. The alpha-helical protein has been studied by the in situ Trp fluorescence quantum yield. This shows that the average distances between coat proteins decrease upon lowering the L/P ratio. In situ cross-linking reactions of the coat protein at high L/P ratios reveal a monomeric state, thus excluding specific aggregation of the coat protein. A monomeric state of detergent-solubilized coat protein is also observed using SDS-PAGE and SDS-HPSEC. On the basis of these results, the smallest in situ aggregational entity of the coat protein is proposed to be a monomer. This finding is discussed in relation to the functional state of the M13 coat protein in the membrane-bound assembly and disassembly processes during infection.
- Published
- 1991
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