359 results on '"Robert Kaptein"'
Search Results
2. Integrative methods in structural biology
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Robert Kaptein and Gerhard Wagner
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Models, Molecular ,Quantitative structure–activity relationship ,Structural biology ,Chemistry ,MEDLINE ,Humans ,Proteins ,Quantitative Structure-Activity Relationship ,Computational biology ,Biology ,Nuclear Magnetic Resonance, Biomolecular ,Biochemistry ,Spectroscopy - Published
- 2019
3. Magnetic field and spin effects on the recombination of radicals on two-dimensional surfaces
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Konstantin L. Ivanov, Vladimir M. Sadovsky, Robert Kaptein, Nikita N. Lukzen, and Renad Z. Sagdeev
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Physics ,Nuclear magnetic resonance ,Condensed matter physics ,Spin polarization ,Radical ,Physical and Theoretical Chemistry ,Spin (physics) ,Recombination ,Magnetic field - Published
- 2013
4. The therapeutic effect of mitochondria-targeted antioxidant SkQ1 and Cistanche deserticola is associated with increased levels of tryptophan and kynurenine in the rat lens
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Olga A. Snytnikova, Robert Kaptein, A. Zh. Fursova, N. A. Stefanova, Nataliya G. Kolosova, Renad Z. Sagdeev, and Yu. P. Tsentalovich
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Cistanche ,Antioxidant ,Plastoquinone ,medicine.medical_treatment ,Cistanche deserticola ,Biophysics ,Pharmacology ,medicine.disease_cause ,Biochemistry ,Antioxidants ,Cataract ,chemistry.chemical_compound ,Lens, Crystalline ,medicine ,Animals ,Rats, Wistar ,Kynurenine ,biology ,Therapeutic effect ,Tryptophan ,General Chemistry ,General Medicine ,biology.organism_classification ,Mitochondria ,Rats ,Treatment Outcome ,medicine.anatomical_structure ,chemistry ,Lens (anatomy) ,Oxidative stress ,Drugs, Chinese Herbal - Abstract
Supplementation of senescence-accelerated OXYS rats with the mitochondria-targeted antioxidant SkQ1 and with the powder from Cistanche deserticola results in the deceleration of the cataract development and even in the improvement of lens transparency. The therapeutic effect of these preparations correlates with a significant elevation of tryptophan and kynurenine levels in the lens. This finding is attributed to a deceleration of the tryptophan and kynurenine oxidation due to antioxidant-assisted reduction of oxidative stress in the lens.
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- 2012
5. Photo-CIDNP 1H-NMR studies of bovine pancreatic phospholipase A2 and its zymogen
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Robert Kaptein, Maarten R. Egmond, and P. J. Hore
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chemistry.chemical_classification ,Phospholipase A ,Enzyme Precursors ,Magnetic Resonance Spectroscopy ,CIDNP ,Chemical shift ,Lasers ,Biophysics ,Nuclear magnetic resonance spectroscopy ,Phospholipase ,Biochemistry ,Phospholipases A ,Crystallography ,Phospholipases A2 ,Enzyme ,chemistry ,Structural Biology ,Phospholipases ,Zymogen ,Proton NMR ,Animals ,Cattle ,Molecular Biology ,Pancreas - Abstract
Bovine pancreatic phospholipase A2 and its zymogen were studied by laser photo-CIDNP 1H-NMR. Resonances of Trp3 and Tyr69 protons of the two proteins were assigned. By varying the delay between a short light pulse and the observation pulse, time dependencies of the CIDNP signals were obtained from which effective T1 values could be derived. The photo-CIDNP chemical shifts, intensities and relaxation data pointed to environmental differences for the Tyr69 residues in the two proteins, while only small differences were noted for the Trp3 residues. The more buried position of Tyr69 in the enzyme relative to the zymogen was related to the ability of the enzyme to bind to micellar aggregates, to which the zymogen is unable to bind.
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- 2016
6. H-1-NMR ASSIGNMENTS AND CONNECTIVITIES OF CYTOSINES IN LAC OPERATOR DNA VIA DOUBLE QUANTUM COHERENCE
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A Volbeda, Robert Kaptein, Ruud M. Scheek, Pj Hore, and Jh Vanboom
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Physics ,chemistry.chemical_compound ,chemistry ,Quantum mechanics ,General Engineering ,lac operon ,DNA ,Double quantum coherence - Published
- 2016
7. Proton nuclear magnetic resonance assignments and surface accessibility of Tryptophan residues in lysozyme using photochemically induced dynamic nuclear polarization spectroscopy
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P. J. Hore and Robert Kaptein
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Magnetic Resonance Spectroscopy ,Light ,Chemistry ,Photochemistry ,Tryptophan ,Nuclear Overhauser effect ,Nuclear magnetic resonance spectroscopy ,Darkness ,Biochemistry ,chemistry.chemical_compound ,Nuclear magnetic resonance ,Solid-state nuclear magnetic resonance ,Egg White ,Proton NMR ,Animals ,Muramidase ,Lysozyme ,Spectroscopy ,Two-dimensional nuclear magnetic resonance spectroscopy ,Chickens - Abstract
Tryptophan resonances in the 360-MHz 1H photochemically induced dynamic nuclear polarization spectrum of hen egg white lysozyme are investigated in detail. All resonances of one tryptophan and six of another are identified and assigned to their respective protons. The methods employed, all involving nuclear spin polarization, include the study of cross-relaxation effects and the use of selective radio-frequency irradiation, Gd3+ as a paramagnetic probe, and riboflavin as the chemically induced dynamic nuclear polarization generating dye. From a comparison of the experimental results with the known X-ray structure of lysozyme, second-stage assignments of the two tryptophan residues (Trp-62 and Trp-123) are proposed. A number of other resonances are characterized, among them Trp-63 C(2)H and four indirectly polarized methyl groups.
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- 2016
8. A PHOTO-CIDNP INVESTIGATION OF TYROSINE MOBILITY AND EXPOSURE IN HUMAN BETA-ENDORPHIN IN THE PRESENCE OF PHOSPHOLIPID MICELLES
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L. Zetta, P. J. Hore, and Robert Kaptein
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Circular dichroism ,CIDNP ,Biophysics ,Phospholipid ,Cell Biology ,Lipid micelle ,Photochemistry ,Biochemistry ,Micelle ,Cross relaxation ,chemistry.chemical_compound ,chemistry ,(NMR) ,Structural Biology ,Photo-CIDNP ,Human β-endorphin ,Genetics ,Organic chemistry ,Tyrosine mobility ,Cross-relaxation ,Tyrosine ,Molecular Biology ,N-dodecylphosphorylcholine - Published
- 2016
9. Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis
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Neil J. Rzechorzek, L. G. Carter, Karl Harlos, Keith S. Wilson, David I. Stuart, Axel Müller, Olga V. Moroz, Robert Kaptein, T. Dierks, Ian W. Boucher, Raymond J. Owens, Rosa Grenha, Robert Esnouf, Elena Blagova, Thomas S. Walter, M. P. Boyle, Nathan R. Zaccai, Vladimir M. Levdikov, Gert E. Folkers, Nick S. Berrow, Mark J. Fogg, N. Milioti, Sarah Sainsbury, Kin Fai Au, Anthony J. Wilkinson, David G. Waterman, Anne K. Kalliomaa, Christoph Meier, and James A. Brannigan
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DNA, Bacterial ,Proteomics ,TRNA modification ,Magnetic Resonance Spectroscopy ,Genetic Vectors ,Target analysis ,Computational biology ,Crystallography, X-Ray ,Structural genomics ,Protein structure ,Bacillus cereus ,Bacterial Proteins ,RNA, Transfer ,Structural Biology ,Escherichia coli ,Cloning, Molecular ,SPINE (molecular biology) ,Spores, Bacterial ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Ligation-independent cloning ,Computational Biology ,General Medicine ,Robotics ,biology.organism_classification ,Bacillus anthracis ,Biochemistry ,Sulfurtransferases ,Crystallization - Abstract
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining `low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
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- 2016
10. Kinetic Study of Propylene Hydrogenation over Pt/Al2O3 by Parahydrogen-Induced Polarization
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Robert Kaptein, Danila A. Barskiy, Igor V. Koptyug, Valery I. Bukhtiyarov, Oleg G. Salnikov, and Kirill V. Kovtunov
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chemistry.chemical_classification ,Order of reaction ,Double bond ,Hydrogen ,Spins ,chemistry.chemical_element ,Kinetic energy ,Spin isomers of hydrogen ,Photochemistry ,Atomic and Molecular Physics, and Optics ,Catalysis ,chemistry ,Physical chemistry ,Polarization (electrochemistry) - Abstract
Parahydrogen-induced polarization has been successfully used for a kinetic study of propylene hydrogenation over a Pt/Al2O3 catalyst. It was shown that the reaction orders with respect to hydrogen are different for the pairwise and the non-pairwise hydrogen addition and are equal to 0.7 and 0.1, respectively. This observation of different reaction orders confirms the coexistence of different types of active sites which are responsible for the overall and the pairwise hydrogen addition to the propylene C=C double bond. Moreover, 0.7 reaction order with respect to H2 for pairwise hydrogen addition indicates that the contribution of pairwise addition depends on the concentration of molecular hydrogen. Therefore, this observation can be developed into a practical tool for producing fluids with highly polarized nuclear spins by changing the hydrogen concentration.
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- 2012
11. Solution Structure and Characterization of the DNA-Binding Activity of the B3BP–Smr Domain
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Rob N. de Jong, Mark Daniëls, Gert E. Folkers, Eiso Ab, Tammo Diercks, Rogier Besseling, and Robert Kaptein
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Models, Molecular ,Magnetic Resonance Spectroscopy ,HMG-box ,Molecular Sequence Data ,Protein domain ,Biology ,Protein Structure, Secondary ,Cell Line ,Evolution, Molecular ,SeqA protein domain ,Structural Biology ,Humans ,Amino Acid Sequence ,B3 domain ,Molecular Biology ,Conserved Sequence ,Genetics ,MutS Proteins ,DNA ,DNA-binding domain ,Endonucleases ,Protein Structure, Tertiary ,Solutions ,DNA Repair Enzymes ,Eukaryotic Cells ,Prokaryotic Cells ,Structural Homology, Protein ,Cyclic nucleotide-binding domain ,Biophysics ,Carrier Proteins ,Hydrophobic and Hydrophilic Interactions ,Sequence Alignment ,Protein Binding ,Binding domain - Abstract
The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer alpha-beta sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.
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- 2008
12. Structure and DNA Binding of the Human Rtf1 Plus3 Domain
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Eiso Ab, Rob N. de Jong, Mark Daniëls, Vincent Truffault, Robert Kaptein, Tammo Diercks, and Gert E. Folkers
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Models, Molecular ,Transcription, Genetic ,HMG-box ,Protein Conformation ,SiRNA binding ,Molecular Sequence Data ,Histone H2B ubiquitination ,DNA, Single-Stranded ,RNA polymerase II ,Biology ,Structural Biology ,Transcription (biology) ,Nucleic Acids ,Suppressor Factors, Immunologic ,Humans ,Amino Acid Sequence ,B3 domain ,Molecular Biology ,Conserved Sequence ,Binding Sites ,DNA ,DNA-binding domain ,Molecular biology ,Cell biology ,biology.protein ,RNA ,Sequence Alignment ,Transcription Factors ,Binding domain - Abstract
SummaryThe yeast Paf1 complex consists of Paf1, Rtf1, Cdc73, Ctr9, and Leo1 and regulates histone H2B ubiquitination, histone H3 methylation, RNA polymerase II carboxy-terminal domain (CTD) Ser2 phosphorylation, and RNA 3′ end processing. We provide structural insight into the Paf1 complex with the NMR structure of the conserved and functionally important Plus3 domain of human Rtf1. A predominantly β-stranded subdomain displays structural similarity to Dicer/Argonaute PAZ domains and to Tudor domains. We further demonstrate that the highly basic Rtf1 Plus3 domain can interact in vitro with single-stranded DNA via residues on the rim of the β sheet, reminiscent of siRNA binding by PAZ domains, but did not detect binding to double-stranded DNA or RNA. We discuss the potential role of Rtf1 Plus3 ssDNA binding during transcription elongation.
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- 2008
13. Analysis of the XPA and ssDNA-binding surfaces on the central domain of human ERCC1 reveals evidence for subfunctionalization
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Chao Zheng, Konstantinos Tripsianes, Jeffrey S. Grinstead, Gert E. Folkers, Robert Kaptein, Rolf Boelens, Devashish Das, NMR-spectroscopie, and Dep Scheikunde
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Models, Molecular ,Protein family ,DNA repair ,Molecular Sequence Data ,DNA, Single-Stranded ,Eukaryotic DNA replication ,DNA-binding protein ,chemistry.chemical_compound ,Structural Biology ,Genetics ,Humans ,Amino Acid Sequence ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Nuclease ,Binding Sites ,biology ,Endonucleases ,Cell biology ,Protein Structure, Tertiary ,Xeroderma Pigmentosum Group A Protein ,DNA-Binding Proteins ,chemistry ,biology.protein ,Subfunctionalization ,Dimerization ,DNA ,Protein Binding - Abstract
Human ERCC1/XPF is a structure-specific endonuclease involved in multiple DNA repair pathways. We present the solution structure of the non-catalytic ERCC1 central domain. Although this domain shows structural homology with the catalytically active XPF nuclease domain, functional investigation reveals a completely distinct function for the ERCC1 central domain by performing interactions with both XPA and single-stranded DNA. These interactions are non-competitive and can occur simultaneously through distinct interaction surfaces. Interestingly, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on a structurally homologous region of the two proteins. Although these regions are strictly conserved in each protein family, amino acid composition and surface characteristics are distinct. We discuss the possibility that after XPF gene duplication, the redundant ERCC1 central domain acquired novel functions, thereby increasing the fidelity of eukaryotic DNA repair.
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- 2007
14. On the role of aromatic side chains in the photoactivation of BLUF domains
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Robert Kaptein, Jeffrey S. Grinstead, Klaas J. Hellingwerf, Magdalena Gauden, Wouter Laan, Rolf Boelens, K.C. Toh, John T. M. Kennis, Ivo H. M. van Stokkum, Rienk van Grondelle, Marcela Avila-Perez, Molecular Microbial Physiology (SILS, FNWI), Biophysics Photosynthesis/Energy, NMR-spectroscopie, and Dep Scheikunde
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Light ,biology ,Photochemistry ,Stereochemistry ,Chemistry ,Wild type ,Electrons ,biology.organism_classification ,Models, Biological ,Biochemistry ,Protein Structure, Secondary ,Protein Structure, Tertiary ,Amino Acids, Aromatic ,Electron transfer ,Rhodobacter sphaeroides ,Intersystem crossing ,Amino Acid Substitution ,Models, Chemical ,FAD binding ,Flavin-Adenine Dinucleotide ,Singlet state ,Protons ,Triplet state ,Nuclear Magnetic Resonance, Biomolecular ,BLUF domain - Abstract
BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides ,i s investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH ¥ -W ¥ radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electron- transfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.
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- 2007
15. NMR studies of membrane proteins
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Robert Kaptein and Gerhard Wagner
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Protein Folding ,Chemistry ,Peripheral membrane protein ,Protein Data Bank (RCSB PDB) ,Membrane Proteins ,Biological membrane ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Article ,Membrane protein ,Structural biology ,Protein–lipid interaction ,Integral membrane protein ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
The functional importance of proteins that interact with biological membranes can hardly be overestimated. About half of the medicinal drug targets are membrane proteins. Nevertheless, the structural biology of these proteins has been very challenging as only a little more than 500 unique membrane protein structures are present in the PDB (out of 100,000 structures) and also accessible in a membrane protein database (http://blanco.biomol.uci.edu/mpstruc/). About 16% of these have been derived by NMR spectroscopy. While NMR is limited by molecular size it has the unparalleled capability of observing internal mobility, which can be analyzed at atomic resolution once resonance assignments are obtained. The main obstacles for assignment, dynamics studies and structure determination are low overexpression levels and a high content of hydrophobic amino acids, necessary for embedding into a biological membrane. Thus, some membrane mimetic must always be part of the protein preparation, both for NMR and for X-ray crystallography. The right choice of the membrane mimetic is crucial and is clearly more difficult than finding optimal conditions for studies of soluble proteins. There are numerous options of conditions to select from, such as kind and mixture of detergents and lipids, their concentration, and the relative protein-to-surfactant ratio.
- Published
- 2015
16. Structural Characterization of Spo0E-like Protein-aspartic Acid Phosphatases That Regulate Sporulation in Bacilli
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Neil J. Rzechorzek, Joanne C. Ladds, James A. Brannigan, Marta Perego, Gert E. Folkers, Vincent Truffault, Eiso Ab, Tammo Diercks, Anthony J. Wilkinson, Rob N. de Jong, Cristina Bongiorni, Keith S. Wilson, Robert Kaptein, Rosa Grenha, and Mark J. Fogg
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DNA, Bacterial ,Models, Molecular ,Amino Acid Motifs ,Molecular Sequence Data ,Phosphatase ,Biology ,Biochemistry ,Protein Structure, Secondary ,Phosphotransferase ,Bacterial Proteins ,Aspartic acid ,Transferase ,Amino Acid Sequence ,Protein Structure, Quaternary ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Spores, Bacterial ,Base Sequence ,Sequence Homology, Amino Acid ,Kinase ,fungi ,Cell Biology ,Phosphoric Monoester Hydrolases ,Recombinant Proteins ,Response regulator ,Genes, Bacterial ,Bacillus anthracis ,Phosphorylation ,Dimerization ,Sporulation in Bacillus subtilis - Abstract
Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.
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- 2006
17. Structural Motifs of Lipid II-Binding Lantibiotics as a Blueprint for Novel Antibiotics
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Eefjan Breukink, Robert Kaptein, and Shang-Te Danny Hsu
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Pharmacology ,Infectious Diseases ,Lipid II ,Biochemistry ,medicine.drug_class ,Antibiotics ,medicine ,Biology ,Lantibiotics ,Structural motif - Published
- 2006
18. Gradual phosphorylation regulates PC4 coactivator function
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Martijn W. H. Pinkse, Hendrik R. A. Jonker, Gert E. Folkers, Robert Kaptein, and Rainer Wechselberger
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inorganic chemicals ,Transcription, Genetic ,Protein Conformation ,Molecular Sequence Data ,DNA, Single-Stranded ,macromolecular substances ,Plasma protein binding ,Biology ,environment and public health ,Biochemistry ,DNA-binding protein ,Phosphorylation cascade ,Serine ,Coactivator ,Amino Acid Sequence ,Phosphorylation ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Transcription factor ,Lysine ,Herpes Simplex Virus Protein Vmw65 ,Cell Biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Biophysics ,Nucleic Acid Conformation ,bacteria ,Casein kinase 2 ,Sequence Alignment ,Protein Binding ,Transcription Factors - Abstract
The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces.
- Published
- 2006
19. The Intrinsically Unstructured Domain of PC4 Modulates the Activity of the Structured Core through Inter- and Intramolecular Interactions
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Gert E. Folkers, Rolf Boelens, Rainer Wechselberger, Robert Kaptein, Hendrik R. A. Jonker, NMR-spectroscopie, Dep Scheikunde, and Sub NMR Spectroscopy
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Models, Molecular ,HMG-box ,Protein Conformation ,DNA, Single-Stranded ,Biology ,Biochemistry ,Cofactor ,Structure-Activity Relationship ,chemistry.chemical_compound ,Humans ,B3 domain ,Nuclear Magnetic Resonance, Biomolecular ,Binding Sites ,General transcription factor ,Lysine ,Herpes Simplex Virus Protein Vmw65 ,DNA ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Crystallography ,chemistry ,Docking (molecular) ,Intramolecular force ,Mutation ,Mutagenesis, Site-Directed ,Trans-Activators ,Biophysics ,biology.protein ,Protein Binding ,Transcription Factors ,Binding domain - Abstract
Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.
- Published
- 2006
20. The Structure of the Human ERCC1/XPF Interaction Domains Reveals a Complementary Role for the Two Proteins in Nucleotide Excision Repair
- Author
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Gert E. Folkers, Hanny Odijk, Konstantinos Tripsianes, Devashish Das, Jan H.J. Hoeijmakers, Robert Kaptein, Rolf Boelens, Eiso Ab, Nicolaas G. J. Jaspers, NMR-spectroscopie, NMR Spectroscopy 1, Dep Scheikunde, and Molecular Genetics
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DNA Repair ,DNA repair ,Complex formation ,Molecular Sequence Data ,Endonuclease ,chemistry.chemical_compound ,Structural Biology ,Hydrolase ,Protein Interaction Mapping ,Humans ,Amino Acid Sequence ,Molecular Biology ,Genetics ,Nuclease ,biology ,Helix-Loop-Helix Motifs ,DNA ,Endonucleases ,Cell biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,chemistry ,International ,biology.protein ,ERCC1 ,Dimerization ,Nucleotide excision repair - Abstract
SummaryThe human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.
- Published
- 2005
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21. The Structure of Bypass of Forespore C, an Intercompartmental Signaling Factor during Sporulation in Bacillus
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Eiso Ab, Anthony J. Wilkinson, Vincent Truffault, Tammo Diercks, James A. Brannigan, Keith S. Wilson, Robert Kaptein, Gert E. Folkers, Rob N. de Jong, Hayley M. Patterson, and Simon M. Cutting
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Models, Molecular ,Protein Folding ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Amino Acid Motifs ,Cell Communication ,Bacillus subtilis ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,RNA polymerase ,Gene expression ,Asymmetric cell division ,Peptide sequence ,Spores, Bacterial ,Genetics ,0303 health sciences ,DNA-Directed RNA Polymerases ,Recombinant Proteins ,Cell biology ,Phenotype ,Electrophoresis, Polyacrylamide Gel ,Protein folding ,Bacterial Outer Membrane Proteins ,Plasmids ,Signal Transduction ,Spectrometry, Mass, Electrospray Ionization ,Cell signaling ,Molecular Sequence Data ,Biology ,Models, Biological ,03 medical and health sciences ,Bacterial Proteins ,Amino Acid Sequence ,Molecular Biology ,030304 developmental biology ,Sequence Homology, Amino Acid ,030306 microbiology ,Cell Membrane ,fungi ,Gene Expression Regulation, Bacterial ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,chemistry ,Mutation ,Gene Deletion ,Transcription Factors - Abstract
Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase sigma-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing sigma(K), (Esigma(K)). sigma(K) is synthesized as an inactive zymogen, pro-sigma(K), which is activated by proteolytic cleavage. Processing of pro-sigma(K) is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-sigma(K) processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-sigma(K) processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded beta-sheet onto one face of which an alpha-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded beta-sheet and three alpha-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the sigma(K) checkpoint are discussed in the light of sequence and structure comparisons.
- Published
- 2005
22. 360-MHz Nuclear Magnetic Resonance and Laser Photochemically Induced Dynamic Nuclear Polarization Studies of Bile Salt Interaction with Porcine Colipase A
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Louis Sarda, Paul Canioni, Patrick J. Cozzone, and Robert Kaptein
- Subjects
Magnetic Resonance Spectroscopy ,Chemical Phenomena ,Swine ,Salt (chemistry) ,Colipase ,Biochemistry ,Micelle ,chemistry.chemical_compound ,Animals ,Molecule ,Organic chemistry ,Histidine ,Colipases ,Lumiflavin ,Micelles ,chemistry.chemical_classification ,Taurodeoxycholic Acid ,biology ,Lasers ,Proteins ,Hydrogen-Ion Concentration ,Chemistry ,Crystallography ,chemistry ,biology.protein ,Proton NMR ,Tyrosine ,Protein folding ,Stoichiometry ,Deoxycholic Acid - Abstract
Porcine pancreatic colipase possesses a particular three-dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343–354; J. Wieloch, B. Borgstrom, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622–1628]. The domain corresponds to the 49–57 and 77–86 β-sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29–42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo-CIDNP) studies of the complexes of colipase with organized bile salt micelles. The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr-I and Tyr-II (Tyr-56 and Tyr-57) and His-II (His-86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo-CIDNP effects of Tyr-I and Tyr-II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219–222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo-CIDNP effects observed in the colipase-micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic. A general model for micelle binding to colipase is proposed and involves a two-step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase.
- Published
- 2005
23. The solution structure of a transient photoreceptor intermediate: Delta 25 photoactive yellow protein
- Author
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Robert Kaptein, Cédric Bernard, Rolf Boelens, Nocky M. Derix, Klaas J. Hellingwerf, David Marks, Klaartje Houben, Michael A. van der Horst, Nico A. J. van Nuland, and Molecular Microbial Physiology (SILS, FNWI)
- Subjects
Models, Molecular ,Protein Folding ,Magnetic Resonance Spectroscopy ,Light ,Protein Conformation ,Beta sheet ,Crystallography, X-Ray ,Photoreceptors, Microbial ,Protein structure ,Bacterial Proteins ,Structural Biology ,Spectroscopy ,Molecular Biology ,Photoactive yellow protein ,Models, Statistical ,Chemistry ,X-Rays ,Halorhodospira halophila ,Nuclear magnetic resonance spectroscopy ,Solution structure ,Protein Structure, Tertiary ,Crystallography ,Models, Chemical ,Protein folding ,Protons ,Alpha helix ,Signal Transduction - Abstract
The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.
- Published
- 2005
24. NMR Study of Mersacidin and Lipid II Interaction in Dodecylphosphocholine Micelles
- Author
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Hans-Georg Sahl, Shang-Te Danny Hsu, Gabriele Bierbaum, Eefjan Breukink, Robert Kaptein, Ben de Kruijff, Alexandre M. J. J. Bonvin, and Nico A. J. van Nuland
- Subjects
Alanine ,chemistry.chemical_classification ,Lipid II ,Chemistry ,Stereochemistry ,Cell Biology ,Lantibiotics ,Biochemistry ,Micelle ,Amino acid ,Molecular dynamics ,Protein structure ,Molecular Biology ,Heteronuclear single quantum coherence spectroscopy - Abstract
Mersacidin belongs to the type B lantibiotics (lanthionine-containing antibiotics) that contain post-translationally modified amino acids and cyclic ring structures. It targets the cell wall precursor lipid II and thereby inhibits cell wall synthesis. In light of the emerging antibiotics resistance problem, the understanding of the antibacterial activity on a structural basis provides a key to circumvent this issue. Here we present solution NMR studies of mersacidin-lipid II interaction in dodecylphosphocholine (DPC) micelles. Distinct solution structures of mersacidin were determined in three different states: in water/methanol solution and in DPC micelles with and without lipid II. The structures in various sample conditions reveal remarkable conformational changes in which the junction between Ala-12 and Abu-13 (where Abu is aminobutyric acid) effectively serves as the hinge for the opening and closure of the ring structures. The DPC micelle-bound form resembles the previously determined NMR and x-ray crystal structures of mersacidin in pure methanol but substantially deviates from the other two states in our current report. The structural changes delineate the large chemical shift perturbations observed during the course of a two-step (15)N-(1)H heteronuclear single quantum coherence titration. They also modulate the surface charge distribution of mersacidin suggesting that electrostatics play a central role in the mersacidin-lipid II interaction. The observed conformational adaptability of mersacidin might be a general feature of lipid II-interacting antibiotics/peptides.
- Published
- 2003
25. Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate
- Author
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Rainer Wechselberger, Nocky M. Derix, Rolf Boelens, Robert Kaptein, Michael A. van der Horst, Nico A. J. van Nuland, Klaas J. Hellingwerf, and Molecular Microbial Physiology (SILS, FNWI)
- Subjects
Protein Folding ,Light ,Photochemistry ,Chemistry ,Glutamine ,Chemical shift ,Mutant ,Glutamic Acid ,Halorhodospira halophila ,Nuclear magnetic resonance spectroscopy ,Glutamic acid ,Chromophore ,Deuterium ,Photoreceptors, Microbial ,Biochemistry ,Crystallography ,Amino Acid Substitution ,Bacterial Proteins ,Mutagenesis, Site-Directed ,Protein folding ,Protons ,Nuclear Magnetic Resonance, Biomolecular - Abstract
The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.
- Published
- 2003
26. X-ray absorption spectroscopic studies of zinc in the N-terminal domain of HIV-2 integrase and model compounds
- Author
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Martinus C. Feiters, Bernt Krebs, Rolf Boelens, Astrid Eijkelenboom, Ronald H.A. Plasterk, Hans Friedrich Nolting, Robert Kaptein, Fusinita M. I. van den Ent, NMR-spectroscopie, NMR Spectroscopy 1, Dep Scheikunde, and Sub NMR Spectroscopy
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Nuclear and High Energy Physics ,X-ray absorption spectroscopy ,Radiation ,Coordination sphere ,Extended X-ray absorption fine structure ,Absorption spectroscopy ,Protein Conformation ,HIV Integrase ,Peptide Fragments ,XANES ,Coordination complex ,Zinc ,chemistry.chemical_compound ,Crystallography ,Absorptiometry, Photon ,chemistry ,K-edge ,HIV-2 ,Imidazole ,Instrumentation ,Physical Organic Chemistry - Abstract
X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the ZnKedge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess β-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboomet al.(1997),Curr. Biol.7, 739–746; (2000),J. Biomol. NMR,18, 119–28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations withEXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 Å (Debye–Waller-type factor 0.009 Å2) and two imidazole ligands with the N atoms at 1.99 Å (Debye–Waller-type factor 0.005 Å2). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn—S and Zn—N distances of 2.3 Å and 2.0 Å, respectively).
- Published
- 2002
27. Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding
- Author
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Rainer Wechselberger, Robert Kaptein, Dawn L. Duval, Kevin D. Augustijn, Peter C. van der Vliet, and Arthur Gutierrez-Hartmann
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Time Factors ,Recombinant Fusion Proteins ,DNA Mutational Analysis ,Mutant ,Response element ,Plasma protein binding ,Biology ,DNA-binding protein ,Proto-Oncogene Protein c-ets-1 ,Proto-Oncogene Proteins ,Animals ,Humans ,Point Mutation ,Phosphorylation ,Transcription factor ,Glutathione Transferase ,Multidisciplinary ,Dose-Response Relationship, Drug ,Proto-Oncogene Proteins c-ets ,Biological Sciences ,Molecular biology ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Kinetics ,embryonic structures ,Biophysics ,Transcription Factor Pit-1 ,Chickens ,Heteronuclear single quantum coherence spectroscopy ,Plasmids ,Protein Binding ,Transcription Factors - Abstract
The POU-domain transcription factor Pit-1 and Ets-1, a member of the ETS family of transcription factors, can associate in solution and synergistically activate the prolactin promoter by binding to a composite response element in the prolactin promoter. We mapped the minimal region of Ets-1 required for the interaction with the Pit-1 POU-homeodomain. Here, we describe a detailed NMR study of the interaction between the POU-homeodomain of Pit-1 and the minimal interacting region of Ets-1. By using heteronuclear single quantum coherence titration experiments, we were able to map exact residues on the POU-homeodomain that are involved in the interaction with this minimal Ets-1 interaction domain. By using our NMR data, we generated point mutants in the POU-homeodomain and tested their effect on the interaction with Ets-1. Our results show that phosphorylation of Pit-1 can regulate the interaction with Ets-1.
- Published
- 2002
28. Analysis of Protein−Carbohydrate Interaction at the Lower Size Limit of the Protein Part (15-Mer Peptide) by NMR Spectroscopy, Electrospray Ionization Mass Spectrometry, and Molecular Modeling
- Author
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Martin Frank, Claus-Wilhelm von der Lieth, René Roy, Rainer Wechselberger, John A. F. Joosten, Robert Kaptein, A. J. R. Heck, Hans-Joachim Gabius, Jasper Kramer, ○ Kate Rittenhouse-Olson, Sabine André, Shan-Yun Lu, Hans-Christian Siebert, and Johannes F.G. Vliegenthart
- Subjects
Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Disaccharide binding ,Protein mass spectrometry ,Stereochemistry ,Electrospray ionization ,Molecular Sequence Data ,Carbohydrates ,Peptide ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,Protein–carbohydrate interactions ,Organic chemistry ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Proteins ,Nuclear magnetic resonance spectroscopy ,Ligand (biochemistry) ,0104 chemical sciences ,chemistry ,Carbohydrate Metabolism - Abstract
Structural analysis of minimally sized lectins will offer insights into fundamentals of intermolecular recognition and potential for biomedical applications. We thus moved significantly beyond the natural limit of lectin size to determine the structure of synthetic mini-lectins in solution, their carbohydrate selectivity and the impact of ligand binding on their conformational behavior. Using three disaccharide (Thomsen-Friedenreich antigen; Gal beta 1,3GalNAc alpha 1,R)-binding pentadecapeptides without internal disulfide bridges as role models, we successfully tested a combined strategy with different techniques of NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling. In solution, the peptides invariably displayed flexibility with rather limited restrictions, shown by NMR experiments including nearly complete resonance assignments and molecular dynamics simulations. The occurrence of aromatic/nonpolar amino acids in the sequence did not lead to formation of a hydrophobic core known from microbial chitinase modules. Selectivity of disaccharide binding was independently observed by mass spectrometry and NMR analysis. Specific ligand interaction yielded characteristic NMR signal alterations but failed to reduce conformational flexibility significantly. We have thereby proven effectiveness of our approach to analyze even low-affinity interactions (not restricted to carbohydrates as ligands). It will be useful to evaluate the impact of rational manipulation of lead peptide sequences.
- Published
- 2002
29. The C Terminus of Apocytochrome b562 Undergoes Fast Motions and Slow Exchange among Ordered Conformations Resembling the Folded State
- Author
-
Michael Czisch, Alexandre M. J. J. Bonvin, Robert Kaptein, Paul D. Barker, and Nicola D'amelio
- Subjects
Protein Folding ,Protein Conformation ,Biochemistry ,Chemical shift index ,Protein structure ,Escherichia coli ,Nuclear Magnetic Resonance, Biomolecular ,Protein secondary structure ,Carbon Isotopes ,Carbon Monoxide ,Nitrogen Isotopes ,Hydrogen bond ,Chemistry ,Chemical shift ,Temperature ,Cytochrome b Group ,Deuterium ,Amides ,Peptide Fragments ,Crystallography ,Helix ,Thermodynamics ,Protein folding ,Hydrogen–deuterium exchange ,Protons ,Apoproteins - Abstract
The present work describes the dynamics of the apo form of cytochrome b(562), a small soluble protein consisting of 106 amino acid residues [Itagaki, E., and Hager, L. P. (1966) J. Biol. Chem. 241, 3687-3695]. The presence of exchange in the millisecond time scale is demonstrated for the last part of helix IV (residues 95-105 in the holo form). The chemical shift index analysis [Wishart, D. S., and Sykes, B. D. (1994) J. Biomol. NMR 4, 171-180] based on H(alpha), C(alpha), C(beta), and C' chemical shifts suggests a larger helical content than shown in the NMR structure based on NOEs. These results indicate the presence of helical-like conformations participating in the exchange process. This hypothesis is consistent with amide deuterium exchange rates and the presence of some hydrogen bonds identified from amide chemical shift temperature coefficients [Baxter, N. J., and Williamson, M. P. (1997) J. Biomol. NMR 9, 359-369]. (15)N relaxation indicates limited mobility for the amide protons of this part of the helix in the picosecond time scale. A 30 ns stochastic dynamics simulation shows small fluctuations around the helical conformation on this time scale. These fluctuations, however, do not result in a significant decrease of the calculated order parameters which are consistent with the experimental (15)N relaxation data. These results resolve an apparent discrepancy in the NMR structures between the disorder observed in helix IV due to a lack of NOEs and the secondary structure predictions based on H(alpha) chemical shifts [Feng, Y., Wand, A. J., and Sligar, S. G. (1994) Struct. Biol. 1, 30-35].
- Published
- 2002
30. Smart management of LTE outdoor site support cabinet-migrate towards simplicity
- Author
-
Robert Kaptein, Oscar Wang, Qi Wei, and Shudong Luo
- Subjects
Engineering ,business.industry ,media_common.quotation_subject ,Cabinet (file format) ,computer.file_format ,Simplicity ,business ,Telecommunications ,computer ,media_common - Published
- 2014
31. The role of level anti-crossings in nuclear spin hyperpolarization
- Author
-
Konstantin L. Ivanov, Andrey N. Pravdivtsev, Robert Kaptein, Hans-Martin Vieth, and Alexandra V. Yurkovskaya
- Subjects
SABRE ,Nuclear and High Energy Physics ,Spin polarization ,CIDNP ,Chemistry ,Spin hyperpolarization ,Polarization (waves) ,Biochemistry ,Induced polarization ,Analytical Chemistry ,Nuclear magnetic resonance ,Materials Science(all) ,Hyperpolarization (physics) ,Magnetization transfer ,Insensitive nuclei enhanced by polarization transfer ,Atomic physics ,Spin (physics) ,Spectroscopy ,Level anti-crossing ,PHIP - Abstract
Nuclear spin hyperpolarization is an important resource for increasing the sensitivity of NMR spectroscopy and MRI. Signal enhancements can be as large as 3–4 orders of magnitude. In hyperpolarization experiments, it is often desirable to transfer the initial polarization to other nuclei of choice, either protons or insensitive nuclei such as 13C and 15N. This situation arises primarily in Chemically Induced Dynamic Nuclear Polarization (CIDNP), Para-Hydrogen Induced Polarization (PHIP), and the related Signal Amplification By Reversible Exchange (SABRE). Here we review the recent literature on polarization transfer mechanisms, in particular focusing on the role of Level Anti-Crossings (LACs) therein. So-called “spontaneous” polarization transfer may occur both at low and high magnetic fields. In addition, transfer of spin polarization can be accomplished by using especially designed pulse sequences. It is now clear that at low field spontaneous polarization transfer is primarily due to coherent spin-state mixing under strong coupling conditions. However, thus far the important role of LACs in this process has not received much attention. At high magnetic field, polarization may be transferred by cross-relaxation effects. Another promising high-field technique is to generate the strong coupling condition by spin locking using strong radio-frequency fields. Here, an analysis of polarization transfer in terms of LACs in the rotating frame is very useful to predict which spin orders are transferred depending on the strength and frequency of the B1 field. Finally, we will examine the role of strong coupling and LACs in magnetic-field dependent nuclear spin relaxation and the related topic of long-lived spin-states.
- Published
- 2014
32. [Untitled]
- Author
-
Alexandre M. J. J. Bonvin, Marc Guenneugues, Klaartje Houben, Robert Kaptein, and Rolf Boelens
- Subjects
Coupling constant ,Quantitative Biology::Biomolecules ,biology ,Chemistry ,Hydrogen bond ,Chemical shift ,Scalar (mathematics) ,Crystal structure ,Dihedral angle ,biology.organism_classification ,Biochemistry ,Crystallography ,Talos ,Beta protein ,Spectroscopy - Abstract
The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein chymotrypsin inhibitor 2. Dihedral angle restraints for the phi and psi angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond 3hbJNC' coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.
- Published
- 2001
33. [Untitled]
- Author
-
Jurgen F. Doreleijers, Mia L. Raves, Keith S. Wilson, Robert Kaptein, Constantin E. Vorgias, and Hans Vis
- Subjects
Crystallography ,Nuclear magnetic resonance ,Chemistry ,HU Protein ,Spin diffusion ,Molecule ,Structure validation ,Nuclear magnetic resonance spectroscopy ,Nuclear magnetic resonance crystallography ,Biochemistry ,Two-dimensional nuclear magnetic resonance spectroscopy ,Spectroscopy ,Spectral line - Abstract
Joint refinement, i.e., the simultaneous refinement of a structure against both nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic data, was performed on the HU protein from Bacillus stearothermophilus (HUBst). The procedure was aimed at investigating the compatibility of the two data sets and at identifying conflicting information. Wherever important differences were found, such as peptide flips in the main-chain conformation, the data were further analyzed to find the cause. The NMR data showed some errors arising either from the manual interpretation of the spectra or from the incorrect account for spin diffusion. The most important artefact inherent to the X-ray data is the crystal packing of the molecules: the effects range from the limitation of the freedom of the flexible parts of the HUBst molecule to possibly one of the peptide flips.
- Published
- 2001
34. Mutations in the glucocorticoid receptor DNA-binding domain mimic an allosteric effect of DNA 1 1Edited by P. E. Wright
- Author
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Jeffrey A. Lefstin, J.-M Teuben, Keith R. Yamamoto, M.A.A van Tilborg, M Kruiskamp, Rolf Boelens, and Robert Kaptein
- Subjects
Transition (genetics) ,Allosteric regulation ,Mutant ,DNA-binding domain ,Biology ,Retinoid X receptor ,chemistry.chemical_compound ,Glucocorticoid receptor ,Biochemistry ,chemistry ,Structural Biology ,Biophysics ,Molecular Biology ,Two-dimensional nuclear magnetic resonance spectroscopy ,DNA - Abstract
Two previously isolated mutations in the glucocorticoid receptor DNA-binding domain (DBD), S459A and P493R, have been postulated to mimic DNA-induced conformational changes in the glucocorticoid receptor DBD, thereby constitutively triggering an allosteric mechanism in which binding of specific DNA normally induces the exposure of otherwise silent glucocorticoid receptor transcriptional activation surfaces. Here we report the three-dimensional structure of the free S459A and P493R mutant DBDs as determined by NMR spectroscopy. The free S459A and P493R structures both display the conformational changes in the DBD dimerization interface that are characteristic of the DNA-bound wild-type DBD, confirming that these mutations mimic an allosteric effect of DNA. A transition between two packing arrangements of the DBD hydrophobic core provides a mechanism for long-range transmission of conformational changes, induced either by the mutations or by DNA binding, to protein-protein contact surfaces.
- Published
- 2000
35. Hydration dynamics of the collagen triple helix by NMR11Edited by P. E. Wright
- Author
-
Murray Goodman, Rolf Boelens, Giuseppe Melacini, Alexandre M. J. J. Bonvin, and Robert Kaptein
- Subjects
Folding (chemistry) ,Crystallography ,Nuclear magnetic resonance ,Aqueous solution ,Structural Biology ,Chemistry ,Collagen helix ,Solvation ,Molecule ,Nanosecond ,Molecular Biology ,Two-dimensional nuclear magnetic resonance spectroscopy ,Triple helix - Abstract
The hydration of the collagen-like Ac-(Gly-Pro-Hyp) 6 -NH 2 triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the first shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a “hopping” hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathrate-like cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen fibers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides.
- Published
- 2000
36. Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solution by NMR: Hydrogen-deuterium exchange data and pH studies
- Author
-
Johnny Hendriks, Robert Kaptein, N.M. Derix, C.J. Craven, Rolf Boelens, Klaas J. Hellingwerf, and Molecular Microbial Physiology (SILS, FNWI)
- Subjects
Protein Folding ,Protein Conformation ,Photochemistry ,Photoreceptors, Microbial ,Biochemistry ,Spectral line ,chemistry.chemical_compound ,Bacterial Proteins ,Amide ,Molecule ,Nuclear Magnetic Resonance, Biomolecular ,Titrimetry ,Halorhodospira halophila ,Nuclear magnetic resonance spectroscopy ,Chromophore ,Hydrogen-Ion Concentration ,Deuterium ,Amides ,Blueshift ,Solutions ,Crystallography ,Dark state ,chemistry ,Thermodynamics ,Hydrogen–deuterium exchange ,Protons - Abstract
The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.
- Published
- 2000
37. NMR investigation of the interaction of the inhibitor protein Im9 with its partner DNase
- Author
-
Michael Czisch, Geoffrey R. Moore, Colin Kleanthous, Ruth Boetzel, Andrew M. Hemmings, Robert Kaptein, and Richard James
- Subjects
Models, Molecular ,Conformational change ,Deoxyribonucleases ,Chemistry ,Escherichia coli Proteins ,Chemical shift ,Molecular Sequence Data ,Colicins ,Inhibitor protein ,Plasma protein binding ,Biochemistry ,Protein Structure, Secondary ,Protein–protein interaction ,NMR spectra database ,Crystallography ,Bacterial Proteins ,Colicin ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Conformational isomerism ,Protein Binding ,Research Article - Abstract
The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13 C and 15 N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3‐84 were 0.76 6 0.14 A for the backbone atoms and 1.366 0.15 A for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 A, respectively, for residues 4‐83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1 H‐ 1 H‐ 13 C NOESY-HSQC spectra with and without 13 C decoupling. The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.
- Published
- 2000
38. [Untitled]
- Author
-
Ronald H.A. Plasterk, Rainer Wechselberger, Rolf Boelens, Astrid Eijkelenboom, Robert Kaptein, and F. M. I. Van Den Ent
- Subjects
biology ,Stereochemistry ,Dimer ,Protein dimer ,Helix-turn-helix ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Integrase ,chemistry.chemical_compound ,Crystallography ,Protein structure ,chemistry ,Helix ,biology.protein ,Salt bridge ,Spectroscopy - Abstract
The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1–55, named IN1−55) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739–746], consists of four α-helices and is well defined. Helices α1, α2 and α3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix α3. The orientation of the two monomeric units with respect to each other shows considerable variation. 15N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN1−55 shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN1−55 [Cai et al. (1997) Nat. Struct. Biol., 4, 567–577], where the corresponding residues are positively charged and neutral ones.
- Published
- 2000
39. [Untitled]
- Author
-
Lucia Banci, Fabio Arnesano, Michael Czisch, Karin van der Wetering, Robert Kaptein, and Ivano Bertini
- Subjects
Magnetic anisotropy ,Paramagnetism ,Condensed matter physics ,Residual dipolar coupling ,Chemistry ,Demagnetizing field ,Anisotropy ,Biochemistry ,Magnetic susceptibility ,Spectroscopy ,Magnetic dipole–dipole interaction ,Magnetic field - Abstract
15N-1H 1J couplings were measured at 500 MHz and 800 MHz for 15N enriched oxidized cytochrome b562 from E. coli. The magnetic field dependence of 70 1J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc). The rdc were used as further constraints to improve the existing structure [Arnesano et al. (1999) Biochemistry, 38, 8657-8670] with a protocol which uses the rhombic anisotropy [Banci et al. (1998) J. Am. Ctherz. Soc., 120, 12903-12909]. The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions.
- Published
- 2000
40. Validation of nuclear magnetic resonance structures of proteins and nucleic acids: Hydrogen geometry and nomenclature
- Author
-
Jurgen F. Doreleijers, Gerrit Vriend, Mia L. Raves, and Robert Kaptein
- Subjects
Chemistry ,Protein Data Bank (RCSB PDB) ,Atom (order theory) ,Geometry ,Protonation ,computer.file_format ,Dihedral angle ,Protein Data Bank ,Biochemistry ,Planarity testing ,Bond length ,Crystallography ,Molecular geometry ,Nuclear magnetic resonance ,Structural Biology ,Molecular Biology ,computer - Abstract
A statistical analysis is reported of 1,200 of the 1,404 nuclear magnetic resonance (NMR)-derived protein and nucleic acid structures deposited in the Protein Data Bank (PDB) before 1999. Excluded from this analysis were the entries not yet fully validated by the PDB and the more than 100 entries that contained < 95% of the expected hydrogens. The aim was to assess the geometry of the hydrogens in the remaining structures and to provide a check on their nomenclature. Deviations in bond lengths, bond angles, improper dihedral angles, and planarity with respect to estimated values were checked. More than 100 entries showed anomalous protonation states for some of their amino acids. Approximately 250,000 (1.7%) atom names differed from the consensus PDB nomenclature. Most of the inconsistencies are due to swapped prochiral labeling. Large deviations from the expected geometry exist for a considerable number of entries, many of which are average structures. The most common causes for these deviations seem to be poor minimization of average structures and an improper balance between force-field constraints for experimental and holonomic data. Some specific geometric outliers are related to the refinement programs used. A number of recommendations for biomolecular databases, modeling programs, and authors submitting biomolecular structures are given.
- Published
- 1999
41. Refined solution structure of the c-terminal DNA-binding domain of human immunovirus-1 integrase
- Author
-
Karl Hård, Ronald H.A. Plasterk, Remco Sprangers, Ramon A. Puras Lutzke, Robert Kaptein, Rolf Boelens, and Astrid Eijkelenboom
- Subjects
biology ,Dimer ,DNA-binding domain ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Homology (biology) ,Integrase ,chemistry.chemical_compound ,Crystallography ,Protein structure ,chemistry ,Structural Biology ,biology.protein ,Molecular Biology ,Root-mean-square deviation ,DNA - Abstract
The structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well-defined dimer interface. The folding topology of the monomer consists of a five-stranded β-barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back-bone root mean square deviation versus the average of 0.46 A. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556–564. © 1999 Wiley-Liss, Inc.
- Published
- 1999
42. [Untitled]
- Author
-
Rolf Boelens, Irene M.A. Nooren, Robert T. Sauer, and Robert Kaptein
- Subjects
Coiled coil ,Leucine zipper ,Dimer ,Repressor ,Nuclear magnetic resonance spectroscopy ,Lac repressor ,Antiparallel (biochemistry) ,Biochemistry ,chemistry.chemical_compound ,Crystallography ,Protein structure ,chemistry ,Structural Biology ,Genetics - Abstract
The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.
- Published
- 1999
43. NMR Experiments for the Study of Photointermediates: Application to the Photoactive Yellow Protein
- Author
-
Geerten W. Vuister, Rolf Boelens, Gilles Rubinstenn, Nico Zwanenburg, Klaas J. Hellingwerf, and Robert Kaptein
- Subjects
Photoactive yellow protein ,Nuclear and High Energy Physics ,Light ,Nitrogen Isotopes ,Chemistry ,Hydrogen bond ,Biophysics ,Hydrogen Bonding ,Photoreceptors, Microbial ,Condensed Matter Physics ,Photochemistry ,Biochemistry ,Isotopes of nitrogen ,Bacterial protein ,Bacterial Proteins ,Nuclear Magnetic Resonance, Biomolecular - Published
- 1999
44. Identification of the Single-stranded DNA Binding Surface of the Transcriptional Coactivator PC4 by NMR
- Author
-
Rolf Boelens, Sebastiaan Werten, Robert Kaptein, Rainer Wechselberger, and Peter C. van der Vliet
- Subjects
Models, Molecular ,Binding Sites ,Magnetic Resonance Spectroscopy ,HMG-box ,Protein Conformation ,Oligonucleotide ,Chemistry ,DNA, Single-Stranded ,Cell Biology ,Biochemistry ,Recombinant Proteins ,DNA-Binding Proteins ,Repressor Proteins ,DNA binding site ,chemistry.chemical_compound ,Heteronuclear molecule ,Trans-Activators ,Biophysics ,Nucleic acid ,Hydrogen–deuterium exchange ,Molecular Biology ,DNA ,Binding domain - Abstract
The C-terminal domain of the eukaryotic transcriptional cofactor PC4 (PC4CTD) is known to bind with nanomolar affinity to single-stranded (ss)DNA. Here, NMR is used to study DNA binding by this domain in more detail. Amide resonance shifts that were observed in a1H15N-HSQC-monitored titration of15N-labeled protein with the oligonucleotide dT18 indicate that binding of the nucleic acid occurs by means of two anti-parallel channels that were previously identified in the PC4CTD crystal structure. The β-sheets and loops that make up these channels exhibit above average flexibility in the absence of ssDNA, which is reflected in higher values of T1ρ, reduced heteronuclear nuclear Overhauser effects and faster deuterium exchange rates for the amides in this region. Upon ssDNA binding, this excess flexibility is significantly reduced. The binding of ssDNA by symmetry-related channels reported here provides a structural rationale for the preference of PC4CTD for juxtaposed single-stranded regions (e.g. in heteroduplexes) observed in earlier work.
- Published
- 1999
45. [Untitled]
- Author
-
Rolf Boelens, Giuseppe Melacini, and Robert Kaptein
- Subjects
symbols.namesake ,Relay ,law ,Chemistry ,Gaussian ,Analytical chemistry ,symbols ,Pulse sequence ,Biochemistry ,Molecular physics ,Spectroscopy ,law.invention - Abstract
A pulse sequence is proposed which uses a train of band-selective pulses for the editing of slow chemical exchange-relay effects in experiments designed to study water-macromolecule interactions. Compared to previous methods, this experiment does not require knowledge of the exact chemical shift of the relaying labile protons and needs only the recording of a single experiment to edit the relay through different exchanging groups resonating at different frequencies. The pulse sequence has been implemented using Gaussian cascades and was applied to the study of the hydration of HEW lysozyme.
- Published
- 1999
46. [Untitled]
- Author
-
Jurgen F. Doreleijers, Ton Rullmann, Robert Kaptein, and Mia L. Raves
- Subjects
Combinatorics ,Protein structure ,Quality assessment ,Statistical analysis ,Completeness (statistics) ,Biochemistry ,Protein secondary structure ,Nmr data ,Spectroscopy ,Mathematics - Abstract
The completeness of experimentally observed NOE restraints of a set of 97 NMR protein structures deposited in the PDB has been assessed. Completeness is defined as the ratio of the number of experimentally observed NOEs and the number of 'expected NOEs'. A practical definition of 'expected NOEs' based on inter-proton distances in the structures up to a given cut-off distance is proposed. The average completeness for the set of 97 structures is 68, 48, and 26% up to 3, 4, and 5 A cut-off distances, respectively. For recent state-of-the-art structures these numbers are approximately 90, 75, and 45%. Almost 20% of the observed NOEs are between atoms that are further than 5 A apart in the final structures. The completeness is independent of the relative surface accessibility and does not depend strongly on residue type, secondary structure or local precision, although the number of observed NOEs in these classes varies considerably. The completeness of NOE restraints is a useful quality criterion in the course of structure refinement. The completeness per residue is more informative than the number of NOEs per residue, which makes it a useful tool to assess the quality of the NMR data set in relation to the resulting structures.
- Published
- 1999
47. [Untitled]
- Author
-
Robert Kaptein, Rolf Boelens, and Giuseppe Melacini
- Subjects
Magnetization ,Chemistry ,Chemical exchange ,Analytical chemistry ,Molecule ,Pulse sequence ,Chymotrypsin inhibitor ,Biological system ,Biochemistry ,Spectroscopy ,Macromolecule - Abstract
A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a √2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15 N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo β-sheet strands were identified, solving previously reported discrepancies between the X-ray and the refined NMR structure of CI2. The proposed experiment is particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand–macromolecule complexes.
- Published
- 1999
48. [Untitled]
- Author
-
Rolf Boelens, Robert Kaptein, A. V. E. George, Robert T. Sauer, and Irene M.A. Nooren
- Subjects
Chemistry ,Hydrogen bond ,Stereochemistry ,Dimer ,Repressor ,Nuclear magnetic resonance spectroscopy ,Antiparallel (biochemistry) ,Biochemistry ,Crystallography ,chemistry.chemical_compound ,Monomer ,Protein structure ,Spectroscopy ,Homotetramer - Abstract
The structure and dynamics of the chymotryptic tetramerization domain of the Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR spectroscopy. Two sets of resonances (A and B) were found, representing the asymmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric NOEs, which were distinguished from the inter-monomeric NOEs by exploiting 13C/15N-filtered NOE experiments, demonstrated a continuous α-helix of approximately seven turns for both the A and B monomers. The asymmetry facilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeric NOEs. The three-dimensional structure revealed an unusual asymmetric packing of a dimer of two antiparallel right-handed intertwined coiled α-helices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amide proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous mutagenesis studies, can be explained by the many hydrophobic and hydrogen bonding interactions that this residue participates in with adjacent monomers.
- Published
- 1999
49. [Untitled]
- Author
-
Paul J.A. van Tilborg, Frans A. A. Mulder, Robert Kaptein, and Rolf Boelens
- Subjects
Zinc finger ,Microsecond ,Crystallography ,Chemical physics ,Chemistry ,Protein dynamics ,Spin echo ,Direct repeat ,Molecule ,DNA-binding domain ,Biochemistry ,Spectroscopy ,Protein tertiary structure - Abstract
Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1ρ experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351–357] were applied to the 9- cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 μs, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271–284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.
- Published
- 1999
50. Solution Structure and Backbone Dynamics of the Photoactive Yellow Protein
- Author
-
Arthur R. Kroon, Gilles Rubinstenn, Petra Düx, Wim Crielaard, Robert Kaptein, Klaas J. Hellingwerf, Geerten W. Vuister, Frans A. A. Mulder, Wouter D. Hoff, Karl Hård, and Rolf Boelens
- Subjects
Models, Molecular ,Protein Folding ,Protein Conformation ,Crystal structure ,Crystallography, X-Ray ,Photoreceptors, Microbial ,Antiparallel (biochemistry) ,Chromatiaceae ,Biochemistry ,law.invention ,Protein structure ,Bacterial Proteins ,law ,Crystallization ,Nuclear Magnetic Resonance, Biomolecular ,Chemistry ,Nuclear magnetic resonance spectroscopy ,Deuterium ,Solutions ,Crystallography ,Picosecond ,Thermodynamics ,Hydrogen–deuterium exchange ,Protein folding ,Protons - Abstract
The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.
- Published
- 1998
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