26 results on '"Garima Jaipuria"'
Search Results
2. New methods for NMR spectral analysis
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Garima Jaipuria, Adil Hayat, and Hanudatta S. Atreya
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Spectral Processing ,Artifact Suppression ,Spatial filtering ,Symmetrization ,Chemistry ,QD1-999 ,Analytical chemistry ,QD71-142 - Abstract
A set of novel methods for signal enhancement and artifact suppression in multidimensional NMR spectra have been developed. These methods, independent of the experiment type, aim at improvising resonance assignments, accurate measurement of NMR parameters and automated peak picking in spectra acquired with low sensitivity or containing artifacts.
- Published
- 2011
3. IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2
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Garima Jaipuria, Divya Shet, Shahid Malik, Monalisa Swain, Hanudatta S. Atreya, Charles A. Galea, Mark G. Slomiany, Steven A. Rosenzweig, Briony E. Forbes, Raymond Norton, and Somnath Mondal
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Intrinsically Disordered Proteins ,Insulin-Like Growth Factor Binding Protein 2 ,Structural Biology ,Humans ,Insulin-Like Growth Factor I ,Molecular Biology ,Biochemistry ,Article ,Peptide Hydrolases ,Protein Binding - Abstract
Functional regulation via conformational dynamics is well known in structured proteins, but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 ( hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L- hIGFBP2) retains its intrinsic disorder upon binding IGF-1 but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.
- Published
- 2022
4. Membrane-embedded TSPO: an NMR view
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Stefan Becker, Garima Jaipuria, Andrei Leonov, Markus Zweckstetter, Karin Giller, Gwladys Rivière, and Loren B. Andreas
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0301 basic medicine ,Magnetic Resonance Spectroscopy ,Biophysics ,Membrane biology ,Solid-state NMR ,metabolism [Cell Membrane] ,03 medical and health sciences ,0302 clinical medicine ,Receptors, GABA ,ddc:570 ,Translocator protein ,medicine ,chemistry [Receptors, GABA] ,Neurodegeneration ,Lipid bilayer ,biology ,Chemistry ,Endoplasmic reticulum ,Cell Membrane ,Structure ,General Medicine ,Lipid ,medicine.disease ,Transmembrane protein ,Dynamics ,Biomarker (cell) ,Cell biology ,metabolism [Receptors, GABA] ,Cholesterol ,030104 developmental biology ,Membrane protein ,biology.protein ,Original Article ,TSPO ,030217 neurology & neurosurgery - Abstract
Translocator Protein (18 kDa) (TSPO) is a mitochondrial transmembrane protein commonly used as a biomarker for neuroinflammation and is also a potential therapeutic target in neurodegenerative diseases. Despite intensive research efforts, the function of TSPO is still largely enigmatic. Deciphering TSPO structure in the native lipid environment is essential to gain insight into its cellular activities and to design improved diagnostic and therapeutic ligands. Here, we discuss the influence of lipid composition on the structure of mammalian TSPO embedded into lipid bilayers on the basis of solid-state NMR experiments. We further highlight that cholesterol can influence both the tertiary and quaternary TSPO structure and also influence TSPO localization in mitochondria-associated endoplasmic reticulum membranes.
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- 2020
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5. Cooperativity of membrane-protein and protein–protein interactions control membrane remodeling by epsin 1 and affects clathrin-mediated endocytosis
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Markus Zweckstetter, King Faisal Yambire, Niels Denkert, Claudia Steinem, Nelli Teske, Daryna Tarasenko, Michael Meinecke, Ira Milosevic, Indrani Mukherjee, Garima Jaipuria, and Benjamin Kroppen
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Phosphatidylinositol 4,5-Diphosphate ,ENTH domain ,Epsin ,Reconstitution of membrane dynamics ,0302 clinical medicine ,epsin ,metabolism [Phosphatidylinositol 4,5-Diphosphate] ,0303 health sciences ,Chemistry ,ultrastructure [Cell Membrane] ,metabolism [Clathrin] ,metabolism [Membrane Lipids] ,Endocytosis ,Clathrin-mediated endocytosis ,genetics [Membrane Proteins] ,Protein Transport ,Membrane ,Membrane curvature ,Molecular Medicine ,Original Article ,genetics [Adaptor Proteins, Vesicular Transport] ,metabolism [Adaptor Proteins, Vesicular Transport] ,Protein Binding ,genetics [Binding Sites] ,chemistry [Cell Membrane] ,metabolism [Cell Membrane] ,Protein–protein interaction ,Membrane Lipids ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Protein Domains ,chemistry [Adaptor Proteins, Vesicular Transport] ,Humans ,chemistry [Membrane Proteins] ,ddc:610 ,Membrane dynamics ,Molecular Biology ,030304 developmental biology ,Pharmacology ,Binding Sites ,Cell Membrane ,Membrane Proteins ,Cell Biology ,Receptor-mediated endocytosis ,chemistry [Membrane Lipids] ,Clathrin ,Microscopy, Electron ,Adaptor Proteins, Vesicular Transport ,Membrane protein ,Mutation ,Biophysics ,metabolism [Membrane Proteins] ,030217 neurology & neurosurgery - Abstract
Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein-induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane have on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo, they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis. Electronic supplementary material The online version of this article (10.1007/s00018-020-03647-z) contains supplementary material, which is available to authorized users.
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- 2020
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6. Cooperativity of membrane-protein and protein-protein interactions control membrane remodeling by epsin 1 and regulate clathrin-mediated endocytosis
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Ira Milosevic, Michael Meinecke, Claudia Steinem, Markus Zweckstetter, King Faisal Yambire, Indrani Murkhejee, Nelli Teske, Niels Denkert, Benjamin Kroppen, Garima Jaipuria, and Daryna Tarasenko
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0303 health sciences ,Epsin ,Chemistry ,Cooperativity ,Receptor-mediated endocytosis ,Endocytosis ,Protein–protein interaction ,03 medical and health sciences ,0302 clinical medicine ,Membrane ,Membrane protein ,Biophysics ,ENTH domain ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane has on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis.
- Published
- 2020
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7. Solid-state NMR investigation of the involvement of the P2 region in tau amyloid fibrils
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Loren B. Andreas, Markus Zweckstetter, Garima Jaipuria, Eckhard Mandelkow, and Adriana Savastano
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0301 basic medicine ,Amyloid ,Protein Conformation ,Protein domain ,Biophysics ,tau Proteins ,Context (language use) ,macromolecular substances ,Intrinsically disordered proteins ,Fibril ,Article ,Epitopes ,03 medical and health sciences ,NMR spectroscopy ,0302 clinical medicine ,Protein structure ,Protein Domains ,mental disorders ,chemistry [Amyloid] ,Humans ,Phosphorylation ,methods [Nuclear Magnetic Resonance, Biomolecular] ,Nuclear Magnetic Resonance, Biomolecular ,metabolism [Amyloid] ,Multidisciplinary ,Chemistry ,chemistry [tau Proteins] ,Proteins ,metabolism [Epitopes] ,metabolism [tau Proteins] ,030104 developmental biology ,Solid-state nuclear magnetic resonance ,ddc:600 ,030217 neurology & neurosurgery - Abstract
The aggregation of hyperphosphorylated tau into amyloid fibrils is closely linked to the progression of Alzheimer’s disease. To gain insight into the link between amyloid structure and disease, the three-dimensional structure of tau fibrils has been studied using solid-state NMR (ssNMR) and cryogenic electron microscopy (cryo-EM). The proline-rich region of tau remains poorly defined in the context of tau amyloid structures, despite the clustering of several phosphorylation sites, which have been associated with Alzheimer’s disease. In order to gain insight into the contribution of the proline-rich region P2 of tau to amyloid fibrils, we studied in vitro aggregated amyloid fibrils of tau constructs, which contain both the proline-rich region P2 and the pseudo-repeats. Using ssNMR we show that the sequence $$^{225}{\text {KVAVVRT}}^{231}$$ 225 KVAVVRT 231 , the most hydrophobic patch within the P2 region, loses its flexibility upon formation of amyloid fibrils. The data suggest a contribution of the P2 region to tau amyloid fibril formation, which might account for some of the unassigned electron density in cryo-EM studies of tau fibrils and could be modulated by tau phosphorylation at the disease-associated AT180 epitope T231/S235.
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- 2020
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8. Crowding interactions perturb structure and stability by destabilizing the stable core of the α-subunit of tryptophan synthase
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Hanudatta S. Atreya, Jagadeesh Gullipalli, Srividya Subramanian, Garima Jaipuria, Rajashekar Varma Kadumuri, and Ramakrishna Vadrevu
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0301 basic medicine ,Protein Denaturation ,Circular dichroism ,Biophysics ,Ficoll ,Cooperativity ,Tryptophan synthase ,010402 general chemistry ,Intrinsically disordered proteins ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,Protein Domains ,Structural Biology ,Enzyme Stability ,Tryptophan Synthase ,Genetics ,Native state ,Humans ,Urea ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,G alpha subunit ,biology ,Chemistry ,Cell Biology ,Crowding ,0104 chemical sciences ,Crystallography ,030104 developmental biology ,biology.protein - Abstract
The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll-70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (αTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of αTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation.
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- 2016
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9. Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials
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Martin J. Scanlon, Bankala Krishnarjuna, Christopher A. MacRaild, Indu R. Chandrashekaran, Hanudatta S. Atreya, Hiromasa Yagi, Shane M. Devine, San Sui Lim, Garima Jaipuria, Raymond S. Norton, Raymond W. Lam, Cael Debono, and Peter J. Scammells
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0301 basic medicine ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Plasmodium falciparum ,Nuclear magnetic resonance spectroscopy ,biology.organism_classification ,Affinities ,Small molecule ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Structural Biology ,parasitic diseases ,biology.protein ,Apical membrane antigen 1 ,Antibody ,Binding site ,Surface plasmon resonance ,Molecular Biology - Abstract
Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright (C) 2016 John Wiley & Sons, Ltd.
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- 2016
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10. Insights into Cholesterol/Membrane Protein Interactions Using Paramagnetic Solid-State NMR
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Stefan Becker, Garima Jaipuria, Markus Zweckstetter, Karin Giller, and Andrei Leonov
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0301 basic medicine ,Catalysis ,DAA 1106 ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Protein structure ,Receptors, GABA ,Acetamides ,Translocator protein ,chemistry [Acetamides] ,Animals ,Amino Acid Sequence ,chemistry [Receptors, GABA] ,Nuclear Magnetic Resonance, Biomolecular ,030102 biochemistry & molecular biology ,biology ,Bzrp protein, mouse ,Chemistry ,Cholesterol ,chemistry [Liposomes] ,Phenyl Ethers ,Organic Chemistry ,Cholesterol binding ,chemistry [Phenyl Ethers] ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,metabolism [Cholesterol] ,Protein Structure, Tertiary ,metabolism [Receptors, GABA] ,030104 developmental biology ,Membrane ,Solid-state nuclear magnetic resonance ,Membrane protein ,metabolism [Acetamides] ,metabolism [Phenyl Ethers] ,ddc:540 ,Liposomes ,biology.protein ,Biophysics ,lipids (amino acids, peptides, and proteins) ,metabolism [Liposomes] ,chemistry [Cholesterol] ,Protein Binding - Abstract
Cholesterol is an essential component of animal cell membranes and impacts the structure and function of membrane proteins. But how cholesterol exerts its functions remains often enigmatic. Here, high-resolution solid-state NMR in combination with paramagnetic cholesterol analogues was shown to be a powerful approach to study the interaction of membrane proteins with cholesterol. Application of the method to the 169-residue translocator protein TSPO provides residue-specific information about its interaction with cholesterol. Comparison with NMR signal perturbations induced by diamagnetic cholesterol furthermore supports changes in the structure of mammalian TSPO caused by cholesterol binding.
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- 2018
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11. Challenges and approaches to understand cholesterol-binding impact on membrane protein function: an NMR view
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Garima Jaipuria, Tina Ukmar-Godec, and Markus Zweckstetter
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0301 basic medicine ,Models, Molecular ,Plasma protein binding ,chemistry [Cell Membrane] ,metabolism [Cell Membrane] ,Cell membrane ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,medicine ,Animals ,Humans ,chemistry [Membrane Proteins] ,ddc:610 ,Binding site ,methods [Nuclear Magnetic Resonance, Biomolecular] ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Pharmacology ,Binding Sites ,030102 biochemistry & molecular biology ,Chemistry ,Cholesterol binding ,Cell Membrane ,Membrane Proteins ,Cell Biology ,metabolism [Cholesterol] ,Transmembrane protein ,Sterol ,030104 developmental biology ,medicine.anatomical_structure ,Cholesterol ,Membrane protein ,analysis [Cholesterol] ,Biophysics ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,metabolism [Membrane Proteins] ,Function (biology) ,Protein Binding - Abstract
Experimental evidence for a direct role of lipids in determining the structure, dynamics, and function of membrane proteins leads to the term 'functional lipids'. In particular, the sterol molecule cholesterol modulates the activity of many membrane proteins. The precise nature of cholesterol-binding sites and the consequences of modulation of local membrane micro-viscosity by cholesterol, however, is often unknown. Here, we review the current knowledge of the interaction of cholesterol with transmembrane proteins, with a special focus on structural aspects of the interaction derived from nuclear magnetic resonance approaches. We highlight examples of the importance of cholesterol modulation of membrane protein function, discuss the specificity of cholesterol binding, and review the proposed binding motifs from a molecular perspective. We conclude with a short perspective on what could be future trends in research efforts targeted towards a better understanding of cholesterol/membrane protein interactions.
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- 2017
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12. Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure
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Rasmus Linser, Markus Zweckstetter, Karin Giller, Suresh K. Vasa, Stefan Becker, Garima Jaipuria, Andrei Leonov, Mariusz Jaremko, and Łukasz Jaremko
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0301 basic medicine ,Magnetic Resonance Spectroscopy ,Science ,Allosteric regulation ,Lipid Bilayers ,General Physics and Astronomy ,General Biochemistry, Genetics and Molecular Biology ,Article ,metabolism [Cell Membrane] ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Protein structure ,Allosteric Regulation ,Receptors, GABA ,Translocator protein ,Animals ,Amino Acid Sequence ,chemistry [Receptors, GABA] ,Protein Structure, Quaternary ,Multidisciplinary ,biology ,Bzrp protein, mouse ,Chemistry ,Cholesterol binding ,Cell Membrane ,General Chemistry ,metabolism [Cholesterol] ,metabolism [Mitochondria] ,Transmembrane protein ,Sterol ,Mitochondria ,Protein Structure, Tertiary ,metabolism [Receptors, GABA] ,030104 developmental biology ,Cholesterol ,Membrane protein ,mitochondrial ,translocator ,protein ,structure ,biology.protein ,Biophysics ,Protein quaternary structure ,ddc:500 ,Protein Multimerization ,metabolism [Lipid Bilayers] ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer–dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSPO's cholesterol-recognition motif leads to structural changes across the protein that shifts the dynamic equilibrium towards the translocator monomer. Consistent with an allosteric mechanism, a mutation within the oligomerization interface perturbs transmembrane regions located up to 35 Å away from the interface, reaching TSPO's cholesterol-binding motif. The lower structural stability of the intervening transmembrane regions provides a mechanistic basis for signal transmission. Our study thus reveals an allosteric signal pathway that connects membrane protein tertiary and quaternary structure with cholesterol binding., The outer mitochondrial membrane translocator protein (TSPO) mediates several mitochondrial functions and binds cholesterol with a high affinity. Here the authors use solid-state NMR to show that cholesterol binding to TSPO results in allosteric changes that modulate TSPO oligomerization.
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- 2017
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13. Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2
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Raymond S. Norton, Bankala Krishnarjuna, Garima Jaipuria, Karyn L. Wilde, Tamir Dingjan, Romain Rouet, Nyssa Drinkwater, Christopher A. MacRaild, Sheena McGowan, Daniel Christ, Jack S. Richards, Jeffrey Seow, Robin F. Anders, Hanudatta S. Atreya, and Rodrigo A.V. Morales
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0301 basic medicine ,Models, Molecular ,medicine.drug_class ,Protein Conformation ,Plasmodium falciparum ,Protozoan Proteins ,Antibodies, Protozoan ,Antigens, Protozoan ,Calorimetry ,Monoclonal antibody ,Crystallography, X-Ray ,Epitope ,03 medical and health sciences ,Epitopes ,Mice ,Antigen ,Structural Biology ,parasitic diseases ,Malaria Vaccines ,medicine ,Animals ,Merozoite surface protein ,Molecular Biology ,biology ,Linear epitope ,Malaria vaccine ,Antibodies, Monoclonal ,Surface Plasmon Resonance ,biology.organism_classification ,Virology ,Molecular biology ,030104 developmental biology ,biology.protein ,Antibody ,Protein Binding - Abstract
Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2(207-222) identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-angstrom crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates. (C) 2017 Elsevier Ltd. All rights reserved.
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- 2016
14. High resolution methyl selective 13C-NMR of proteins in solution and solid state
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Garima Jaipuria, Nitin P. Lobo, Hanudatta S. Atreya, and Divya Shet
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Chemistry ,Physics ,Resolution (electron density) ,NMR Research Centre (Formerly Sophisticated Instruments Facility) ,Analytical chemistry ,Solid-state ,Solid State & Structural Chemistry Unit ,Biochemistry ,Paramagnetism ,Crystallography ,medicine.anatomical_structure ,Solid-state nuclear magnetic resonance ,medicine ,Molecule ,Selectivity ,Spin (physics) ,Nucleus ,Spectroscopy - Abstract
New C-13-detected NMR experiments have been devised for molecules in solution and solid state, which provide chemical shift correlations of methyl groups with high resolution, selectivity and sensitivity. The experiments achieve selective methyl detection by exploiting the one bond J-coupling between the C-13-methyl nucleus and its directly attached C-13 spin in a molecule. In proteins such correlations edit the C-13-resonances of different methyl containing residues into distinct spectral regions yielding a high resolution spectrum. This has a range of applications as exemplified for different systems such as large proteins, intrinsically disordered polypeptides and proteins with a paramagnetic centre.
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- 2012
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15. Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH
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Garima Jaipuria, Kousik Chandra, Hanudatta S. Atreya, and Divya Shet
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chemistry.chemical_classification ,Models, Statistical ,biology ,Ubiquitin ,Stereochemistry ,Chemistry ,Chemical shift ,Proteins ,High resolution ,Biochemistry ,Maltose-Binding Proteins ,Recombinant Proteins ,Amino acid ,Insulin-Like Growth Factor Binding Proteins ,Maltose-binding protein ,Protein structure ,Computational chemistry ,Escherichia coli ,biology.protein ,Amino Acids ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Curse of dimensionality - Abstract
We present reduced dimensionality (RD) 3D HN(CA)NH for efficient sequential assignment in proteins. The experiment correlates the (15)N and (1)H chemical shift of a residue ('i') with those of its immediate N-terminal (i - 1) and C-terminal (i + 1) neighbors and provides four-dimensional chemical shift correlations rapidly with high resolution. An assignment strategy is presented which combines the correlations observed in this experiment with amino acid type information obtained from 3D CBCA(CO)NH. By classifying the 20 amino acid types into seven distinct categories based on (13)C(β) chemical shifts, it is observed that a stretch of five sequentially connected residues is sufficient to map uniquely on to the polypeptide for sequence specific resonance assignments. This method is exemplified by application to three different systems: maltose binding protein (42 kDa), intrinsically disordered domain of insulin-like growth factor binding protein-2 and Ubiquitin. Fast data acquisition is demonstrated using longitudinal (1)H relaxation optimization. Overall, 3D HN(CA)NH is a powerful tool for high throughput resonance assignment, in particular for unfolded or intrinsically disordered polypeptides.
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- 2012
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16. New methods for NMR spectral analysis
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Hanudatta S. Atreya, Adil Hayat, and Garima Jaipuria
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Spatial filtering ,lcsh:QD71-142 ,Chemistry ,Artifact Suppression ,lcsh:Analytical chemistry ,Symmetrization ,General Physics and Astronomy ,Resonance ,General Chemistry ,General Biochemistry, Genetics and Molecular Biology ,Spectral line ,lcsh:Chemistry ,NMR spectra database ,Signal enhancement ,Spectral Processing ,Nuclear magnetic resonance ,lcsh:QD1-999 ,Artifact suppression ,Peak picking ,General Materials Science ,Spectral analysis ,Sensitivity (control systems) ,General Environmental Science - Abstract
A set of novel methods for signal enhancement and artifact suppression in multidimensional NMR spectra have been developed. These methods, independent of the experiment type, aim at improvising resonance assignments, accurate measurement of NMR parameters and automated peak picking in spectra acquired with low sensitivity or containing artifacts.
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- 2011
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17. Spontaneous self-assembly of designed cyclic dipeptide (Phg-Phg) into two-dimensional nano- and mesosheets
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M. Pandeeswar, Garima Jaipuria, Thimmaiah Govindaraju, Hanudatta S. Atreya, and Kolleboyina Jayaramulu
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Thermogravimetric analysis ,chemistry.chemical_compound ,Crystallography ,Morphology (linguistics) ,Dipeptide ,Lateral surface ,Chemistry ,Nano ,Stacking ,Thermal stability ,General Chemistry ,Self-assembly - Abstract
In this study, we present the spontaneous self-assembly of designed simplest aromatic cyclic dipeptides of (l-Phg-l-Phg) and (d-Phg-l-Phg) to form highly stable two-dimensional (2D) nano- and mesosheets with large lateral surface area. Various microscopy data revealed that the morphology of 2D mesosheets resembles the hierarchical natural materials with layered structure. Solution and solid-state NMR studies on cyclo(l-Phg-l-Phg) revealed the presence of strong (N–H–O) hydrogen-bonded molecular chains supported by aromatic π–π interactions to form 2D mesosheets. Interestingly, cyclo(d-Phg-l-Phg) self-assembles to form single-crystalline as well as non-crystalline 2D rhomboid sheets with large lateral dimension. X-ray diffraction analysis revealed the stacking of (N–H–O) hydrogen-bonded molecular layers along c-axis supported by aromatic π–π interactions. The thermogravimetric analysis shows two transitions with overall high thermal stability attributed to layered hierarchy found in 2D mesosheets.
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- 2011
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18. Amino acid selective unlabeling for sequence specific resonance assignments in proteins
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Garima Jaipuria, Patrick D'Silva, Bankala Krishnarjuna, Hanudatta S. Atreya, and Anushikha Thakur
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Saccharomyces cerevisiae Proteins ,Sequence specific resonance assignment ,Stereochemistry ,Molecular Sequence Data ,Polypeptide chain ,010402 general chemistry ,Electronic Supplementary Material ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,Protein structure ,Mitochondrial Precursor Protein Import Complex Proteins ,Selective unlabeling ,Amino Acid Sequence ,Amino Acids ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,Spectroscopy ,030304 developmental biology ,chemistry.chemical_classification ,Carbon Isotopes ,0303 health sciences ,Nitrogen Isotopes ,Ubiquitin ,Chemistry ,Chemical shift ,Membrane Transport Proteins ,Proteins ,Resonance ,0104 chemical sciences ,Amino acid ,Isotope Labeling ,Heteronuclear single quantum coherence spectroscopy - Abstract
Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly 13C/15N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {12COi–15Ni+1}-filtered HSQC, which aids in linking the 1HN/15N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to 2H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of 14N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies. Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9459-z) contains supplementary material, which is available to authorized users.
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- 2010
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19. High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination
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Anushikha Thakur, Garima Jaipuria, Patrick D'Silva, and Hanudatta S. Atreya
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Distance constraints ,Molecular Sequence Data ,High resolution ,Methylation ,Biochemistry ,symbols.namesake ,Protein structure ,Leucine ,Escherichia coli ,Humans ,Amino Acid Sequence ,Isoleucine ,Protein Structure, Quaternary ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Carbon Isotopes ,Fourier Analysis ,Chemistry ,Chemical shift ,Resolution (electron density) ,NMR Research Centre (Formerly Sophisticated Instruments Facility) ,Proteins ,Solid State & Structural Chemistry Unit ,Resonance ,Valine ,Deuterium ,Crystallography ,Fourier transform ,symbols ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of C-13 and H-1 chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing of methyl groups into distinct spectral regions by combining their C-13 chemical shifts with that of the neighboring, directly attached, C-13 nucleus. Using this principle, we present three GFT experiments: (a) (4,3)D NOESY-HCCH, (b) (4,3)D H-1-TOCSY-HCCH and (c) (4,3)D C-13-TOCSY-HCCH. These experiments provide unique 4D spectral information rapidly with high sensitivity and resolution for side-chain resonance assignments and NOE analysis of methyl groups. This is exemplified by (4,3)D NOESY-HCCH data acquired for 17.9 kDa non-deuterated cytosolic human J-protein co-chaperone, which provided crucial long-range distance constraints for its 3D structure determination.
- Published
- 2010
- Full Text
- View/download PDF
20. Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling
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Abhinav Dubey, Hanudatta S. Atreya, Garima Jaipuria, Rajashekar Varma Kadumuri, and Ramakrishna Vadrevu
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0301 basic medicine ,Stereochemistry ,Molecular Sequence Data ,Tripeptide ,010402 general chemistry ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,Triple-resonance nuclear magnetic resonance spectroscopy ,Escherichia coli ,Tryptophan Synthase ,Humans ,Amino Acid Sequence ,Amino Acids ,Molecular Biology ,Peptide sequence ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Nitrogen Isotopes ,Organic Chemistry ,Proteins ,Nuclear magnetic resonance spectroscopy ,Deuterium ,0104 chemical sciences ,Amino acid ,Insulin-Like Growth Factor Binding Protein 2 ,030104 developmental biology ,Structural biology ,chemistry ,Molecular Medicine ,Two-dimensional nuclear magnetic resonance spectroscopy ,Heteronuclear single quantum coherence spectroscopy - Abstract
A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D N-15,H-1]HSQC spectrum with two 2D spectra can result in approximate to 50% assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12kDa) and the 29kDa (268 residue) -subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions, even prior to complete resonance assignments.
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- 2015
21. Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials
- Author
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Bankala, Krishnarjuna, San Sui, Lim, Shane M, Devine, Cael O, Debono, Raymond, Lam, Indu R, Chandrashekaran, Garima, Jaipuria, Hiromasa, Yagi, Hanudatta S, Atreya, Martin J, Scanlon, Christopher A, MacRaild, Peter J, Scammells, and Raymond S, Norton
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Models, Molecular ,Binding Sites ,Protein Conformation ,Plasmodium falciparum ,Protozoan Proteins ,Membrane Proteins ,Antigens, Protozoan ,Small Molecule Libraries ,Antimalarials ,Thiazoles ,Drug Design ,Humans ,Pyrazoles ,Benzimidazoles ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Protein Binding - Abstract
Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John WileySons, Ltd.
- Published
- 2015
22. Structure of the mammalian TSPO/PBR protein
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Mariusz Jaremko, Stefan Becker, Garima Jaipuria, Markus Zweckstetter, and Łukasz Jaremko
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Models, Molecular ,chemistry [Bacterial Proteins] ,metabolism [Bacterial Proteins] ,Endogeny ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Bacterial Proteins ,Receptors, GABA ,Translocator protein ,Animals ,Humans ,Binding site ,chemistry [Receptors, GABA] ,Receptor ,030304 developmental biology ,Mammals ,0303 health sciences ,Binding Sites ,biology ,Bzrp protein, mouse ,metabolism [Mammals] ,Ligand (biochemistry) ,Isoquinolines ,Transmembrane protein ,3. Good health ,metabolism [Receptors, GABA] ,ddc:540 ,PK 11195 ,biology.protein ,pharmacology [Isoquinolines] ,030217 neurology & neurosurgery ,Synthetic ligands ,Function (biology) ,drug effects [Binding Sites] - Abstract
The 3D structure of the 18-kDa transmembrane (TM) protein TSPO (translocator protein)/PBR (peripheral benzodiazepine receptor), which contains a binding site for benzodiazepines, is important to better understand its function and regulation by endogenous and synthetic ligands. We have recently determined the structure of mammalian TSPO/PBR in complex with the diagnostic ligand PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; Jaremko et al. (2014) Science 343, 1363–1366], providing for the first time atomic-level insight into the conformation of this protein, which is up-regulated in various pathological conditions including Alzheimer's disease and Parkinson's disease. Here, we review the studies which have probed the structural properties of mammalian TSPO/PBR as well as the homologues bacterial tryptophan-rich sensory proteins (TspOs) over the years and provide detailed insight into the 3D structure of mouse TSPO (mTSPO)/PBR in complex with PK11195.
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- 2015
- Full Text
- View/download PDF
23. A fast NMR method for resonance assignments: application to metabolomics
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U. Shubhashree, Detlef Moskau, Garima Jaipuria, Abhinav Dubey, Satish Kumar Adiga, Hanudatta S. Atreya, and Shivanand Pudakalakatti
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Carbon Isotopes ,Magnetic Resonance Spectroscopy ,Fourier Analysis ,Analytical chemistry ,NMR Research Centre (Formerly Sophisticated Instruments Facility) ,Solid State & Structural Chemistry Unit ,Covariance ,Biochemistry ,NMR spectra database ,symbols.namesake ,Data acquisition ,Fourier transform ,Metabolomics ,symbols ,Projection (set theory) ,Biological system ,Spectroscopy ,Peptides ,Nuclear Magnetic Resonance, Biomolecular ,Mathematics ,Curse of dimensionality - Abstract
We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled ((12)C) or (13)C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [(13)C, (1)H] HSQC-TOCSY, (2) 2D (1)H-(1)H TOCSY and (3) 2D (13)C-(1)H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [(13)C, (1)H] HSQC-TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete (1)H and (13)C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.
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- 2014
24. Amino acid selective labeling and unlabeling for protein resonance assignments
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Garima, Jaipuria, B, Krishnarjuna, Somnath, Mondal, Abhinav, Dubey, and Hanudatta S, Atreya
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Isotope Labeling ,Molecular Sequence Data ,Proteins ,Amino Acid Sequence ,Amino Acids ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Structural characterization of proteins by NMR spectroscopy begins with the process of sequence specific resonance assignments in which the (1)H, (13)C and (15)N chemical shifts of all backbone and side-chain nuclei in the polypeptide are assigned. This process requires different isotope labeled forms of the protein together with specific experiments for establishing the sequential connectivity between the neighboring amino acid residues. In the case of spectral overlap, it is useful to identify spin systems corresponding to the different amino acid types selectively. With isotope labeling this can be achieved in two ways: (i) amino acid selective labeling or (ii) amino acid selective 'unlabeling'. This chapter describes both these methods with more emphasis on selective unlabeling describing the various practical aspects. The recent developments involving combinatorial selective labeling and unlabeling are also discussed.
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- 2012
25. GFT projection NMR for efficient (1)H/ (13)C sugar spin system identification in nucleic acids
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Garima Jaipuria, Bharathwaj Sathyamoorthy, Victor Beaumont, Thomas Szyperski, Gabriele Varani, and Hanudatta S. Atreya
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chemistry.chemical_classification ,Carbon Isotopes ,Fourier Analysis ,Chemistry ,Stereochemistry ,Carbon-13 ,Analytical chemistry ,Resonance (chemistry) ,Deuterium ,Biochemistry ,Spectral line ,Ribonucleoprotein, U1 Small Nuclear ,NMR spectra database ,symbols.namesake ,Fourier transform ,Nucleic Acids ,symbols ,Nucleic acid ,RNA Precursors ,Humans ,Nucleotide ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify (1)H/(13)C sugar spin systems in (13)C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of (13)C-(1)H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein.
- Published
- 2012
26. High resolution methyl selective ¹³C-NMR of proteins in solution and solid state
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Garima, Jaipuria, Nitin Prakash, Lobo, Divya, Shet, and Hanudatta S, Atreya
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Solutions ,Carbon Isotopes ,Proteins ,Peptides ,Nuclear Magnetic Resonance, Biomolecular - Abstract
New ¹³C-detected NMR experiments have been devised for molecules in solution and solid state, which provide chemical shift correlations of methyl groups with high resolution, selectivity and sensitivity. The experiments achieve selective methyl detection by exploiting the one bond J-coupling between the ¹³C-methyl nucleus and its directly attached ¹³C spin in a molecule. In proteins such correlations edit the ¹³C-resonances of different methyl containing residues into distinct spectral regions yielding a high resolution spectrum. This has a range of applications as exemplified for different systems such as large proteins, intrinsically disordered polypeptides and proteins with a paramagnetic centre.
- Published
- 2012
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