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Kurt Wüthrich - One of the best experts on this subject based on the ideXlab platform.
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Nuclear Magnetic Resonance Spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL
Protein Science, 2011Co-Authors: Eda Koculi, Arthur L. Horwich, Reto Horst, Kurt WüthrichAbstract:Nuclear magnetic resonance (NMR) observation of the uniformly 2H,15N-labeled stringent 33-kDa substrate protein rhodanese in a productive complex with the uniformly 14N-labeled 400 kDa single-ring version of the E. coli chaperonin GroEL, SR1, was achieved with the use of Transverse Relaxation-Optimized Spectroscopy, cross-correlated relaxation-induced polarization transfer, and cross-correlated relaxation-enhanced polarization transfer. To characterize the NMR-observable parts of the bound rhodanese, coherence buildup rates by different magnetization transfer mechanisms were measured, and effects of covalent crosslinking of the rhodanese to the apical binding surface of SR1 were investigated. The results indicate that the NMR-observable parts of the SR1-bound rhodanese are involved in intracomplex rate processes, which are not related to binding and release of the substrate protein from the SR1 binding surface. Rather, they correspond to mobility of the stably bound substrate, which thus appears to include flexibly disordered polypeptide segments devoid of long-lived secondary structures or tertiary folds, as was previously observed also with the smaller substrate human dihydrofolate reductase.
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NMR characterization of membrane protein-detergent micelle solutions by use of microcoil equipment.
Journal of the American Chemical Society, 2009Co-Authors: Pawel Stanczak, Reto Horst, Pedro Serrano, Kurt WüthrichAbstract:Using microcoil NMR technology, the uniformly 2H,15N-labeled integral membrane protein OmpX, and the phosphocholine derivative detergent Fos-10 (n-decylphosphocholine), we investigated solutions of mixed protein−detergent micelles to determine the influence of the detergent concentration on the NMR spectra of the protein. In a first step, we identified key parameters that influence the composition of the micelle solutions, which resulted in a new protocol for the preparation of well-defined concentrated protein solutions. This led to the observation that high-quality 2D [15N,1H]-Transverse Relaxation-Optimized Spectroscopy (TROSY) spectra of OmpX reconstituted in mixed micelles with Fos-10 were obtained only in a limited range of detergent concentrations. Outside of this range from about 90−180 mM, we observed a significant decrease of the average peak intensity. Relaxation-Optimized NMR measurements of the rotational and translational diffusion coefficients of the OmpX/Fos-10 mixed micelles, Dr and Dt, r...
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Protein structures in solution: (1) effects of solvation; (2) studies of large molecules
AIP Conference Proceedings, 2008Co-Authors: Kurt WüthrichAbstract:The general theme of this presentation is nuclear magnetic resonance (NMR) Spectroscopy in structural biology. Two aspects of special interest to biological physicists are discussed. These are, firstly, the results of NMR studies on the solvation of proteins and nucleic acids in solution and, secondly, the physical foundations of a novel NMR approach for studies with very large particles in solution, Transverse Relaxation-Optimized Spectroscopy (TROSY).
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Transverse Relaxation-Optimized NMR Spectroscopy with Biomacromolecular Structures in Solution
Encyclopedia of Magnetic Resonance, 2007Co-Authors: Kurt Wüthrich, Gerhard WiderAbstract:The sections in this article are 1 Introduction 2 The Foundations of Transverse Relaxation-Optimized Spectroscopy (TROSY) 3 TROSY in NMR Experiments with Biological Macromolecules 4 New Stable Isotope Labeling Strategies for TROSY NMR with Large Structures 5 Combining TROSY with Cross Correlated Relaxation-Induced Polarization Transfer for Studies of Very Large Structures 6 Conclusions and Outlook 7 Acknowledgements 8 Biographical Sketches Related Articles
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Proton–proton Overhauser NMR Spectroscopy with polypeptide chains in large structures
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Reto Horst, Gerhard Wider, Jocelyne Fiaux, Eric B. Bertelsen, Arthur L. Horwich, Kurt WüthrichAbstract:The use of 1H–1H nuclear Overhauser effects (NOE) for structural studies of uniformly deuterated polypeptide chains in large structures is investigated by model calculations and NMR experiments. Detailed analysis of the evolution of the magnetization during 1H–1H NOE experiments under slow-motion conditions shows that the maximal 1H–1H NOE transfer is independent of the overall rotational correlation time, even in the presence of chemical exchange with the bulk water, provided that the mixing time is adjusted for the size of the structure studied. 1H–1H NOE buildup measurements were performed for the 472-kDa complex of the 72-kDa cochaperonin GroES with a 400-kDa single-ring variant of the chaperonin GroEL (SR1). These experiments demonstrate that multidimensional NOESY experiments with cross-correlated relaxation-enhanced polarization transfer and Transverse Relaxation-Optimized Spectroscopy elements can be applied to structures of molecular masses up to several hundred kilodaltabs, which opens new possibilities for studying functional interactions in large maromolecular assemblies in solution.
Konstantin Pervushin - One of the best experts on this subject based on the ideXlab platform.
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Identification of a polyoxometalate inhibitor of the DNA binding activity of Sox2.
ACS Chemical Biology, 2011Co-Authors: Kamesh Narasimhan, Konstantin Pervushin, Shubhadra Pillay, Nor Rizal Bin Ahmad, Zsolt Bikádi, Eszter Hazai, Prasanna R. Kolatkar, Ralf JauchAbstract:Aberrant expression of transcription factors is a frequent cause of disease, yet drugs that modulate transcription factor protein−DNA interactions are presently unavailable. To this end, the chemical tractability of the DNA binding domain of the stem cell inducer and oncogene Sox2 was explored in a high-throughput fluorescence anisotropy screen. The screening revealed a Dawson polyoxometalate (K6[P2Mo18O62]) as a direct and nanomolar inhibitor of the DNA binding activity of Sox2. The Dawson polyoxometalate (Dawson-POM) was found to be selective for Sox2 and related Sox-HMG family members when compared to unrelated paired and zinc finger DNA binding domains. [15N,1H]-Transverse relaxation optimized Spectroscopy (TROSY) experiments coupled with docking studies suggest an interaction site of the POM on the Sox2 surface that enabled the rationalization of its inhibitory activity. The unconventional molecular scaffold of the Dawson-POM and its inhibitory mode provides strategies for the development of drugs th...
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Investigation of ligand binding and protein dynamics in Bacillus subtilis chorismate mutase by Transverse relaxation optimized Spectroscopy-nuclear magnetic resonance.
Biochemistry, 2005Co-Authors: Alexander Eletsky, Alexander Kienhöfer, Donald Hilvert, Konstantin PervushinAbstract:The structural and dynamical consequences of ligand binding to a monofunctional chorismate mutase from Bacillus subtilis have been investigated by solution NMR Spectroscopy. TROSY methods were employed to assign 98% of the backbone 1HN, 1Hα, 15N, 13C‘, and 13Cα resonances as well as 86% of the side chain 13C resonances of the 44 kDa trimeric enzyme at 20 °C. This information was used to map chemical shift perturbations and changes in intramolecular mobility caused by binding of prephenate or a transition state analogue to the X-ray structure. Model-free interpretation of backbone dynamics for the free enzyme and its complexes based on 15N relaxation data measured at 600 and 900 MHz showed significant structural consolidation of the protein in the presence of a bound ligand. In agreement with earlier structural and biochemical studies, substantial ordering of 10 otherwise highly flexible residues at the C-terminus is particularly notable. The observed changes suggest direct contact between this protein seg...
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Transverse Relaxation Optimized Spectroscopy
Protein NMR for the Millennium, 2002Co-Authors: Konstantin PervushinAbstract:TROSY represents a spectroscopic contribution in a constant pursuit of NMR structures of ever larger proteins, nucleic acids and their complexes. As it was theoretically predicted and proven in practice the most efficient application of TROSY is achieved in combination with the selective or uniform deuteration of biomolecules, in particular proteins. In this way biomolecular particles with the molecular sizes exceeding 100 kDa have now become amenable for high resolution NMR studies. Due to its simplicity and flexibility the TROSY principle has also been integrated into many other NMR applications not necessary targeting extremely large biomolecues. TROSY-based NMR experiments have been developed for the detection and characterization of weak interactions such as scalar couplings across hydrogen bonds and residual dipolar couplings. The use of TROSY in Nuclear Overhauser Spectroscopy might significantly facilitate the spectral analysis by suppressing the strong diagonal peaks benefiting studies of a wide range of biomolecules.
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Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli
FEBS Letters, 2001Co-Authors: César Fernández, Konstantin Pervushin, Christian Hilty, Koba Adeishvili, S. Bonjour, Kurt WüthrichAbstract:Abstract Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (Transverse Relaxation-Optimized Spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA β-barrel from those of OmpX. Overall, the present data demonstrate that Relaxation-Optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.
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[13C,13C]- and [13C,1H]-TROSY in a Triple Resonance Experiment for Ribose−Base and Intrabase Correlations in Nucleic Acids1
Journal of the American Chemical Society, 2001Co-Authors: Roland Riek, Konstantin Pervushin, César Fernández, Masatsune Kainosho, Kurt WüthrichAbstract:A novel TROSY (Transverse relaxation−optimized Spectroscopy) element is introduced that exploits cross-correlation effects between 13C−13C dipole−dipole (DD) coupling and 13C chemical shift anisotropy (CSA) of aromatic ring carbons. Although these 13C−13C effects are smaller than the previously described [13C,1H]-TROSY effects for aromatic 13C−1H moieties, their constructive use resulted in further Transverse relaxation−optimization by up to 15% for the resonances in a 17 kDa protein−DNA complex. As a practical application, two- and three-dimensional versions of the HCN triple resonance experiment for obtaining ribose−base and intrabase correlations in the nucleotides of DNA and RNA (Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. J. Biomol. NMR 1993, 3, 721−727) have been implemented with [13C,1H]- and [13C,13C]-TROSY elements to reduce the rate of Transverse relaxation during the polarization transfers between ribose 13C1‘ and base 15N1/9 spins, and between 13C6/8 and N1/9 within the bases. The...
Bernd Reif - One of the best experts on this subject based on the ideXlab platform.
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TROSY effects in MAS solid-state NMR
Concepts in Magnetic Resonance Part A, 2008Co-Authors: Veniamin Chevelkov, Bernd ReifAbstract:Use of Transverse Relaxation-Optimized Spectroscopy (TROSY) type techniques had a dramatic impact on the study of large proteins with a molecular weight >30kDa for solution-state NMR. In the solid-state, such an effect would not be expected a prior, as the investigated molecules are immobilized. However, local motions induce fluctuations of the local fields experienced by the nuclear spins and, this way, are effective for relaxation. We demonstrate that protein dynamics can significantly influence the resonance line width in ultra high resolution MAS (magic angle spinning) solid-state NMR experiments. Averaging of the 15NHα/β multiplet components as a consequence of 1H decoupling induces effective broadening of the 15N resonance. Application of TROSY type techniques that select only the narrow component of the multiplet pattern results in an increased resolution and, thus, will be of benefit for MAS solid-state NMR Spectroscopy. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 143–156, 2008.
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TROSY effects in MAS solid-state NMR
Concepts in Magnetic Resonance Part A: Bridging Education and Research, 2008Co-Authors: Veniamin Chevelkov, Bernd ReifAbstract:Use of Transverse Relaxation-Optimized Spectroscopy (TROSY) type techniques had a dramatic impact on the study of large proteins with a molecular weight >30kDa for solution-state NMR. In the solid-state, such an effect would not be expected a prior, as the investigated molecules are immobilized. However, local motions induce fluctuations of the local fields experienced by the nuclear spins and, this way, are effective for relaxation. We demonstrate that protein dynamics can significantly influence the resonance line width in ultra high resolution MAS (magic angle spinning) solid-state NMR experiments. Averaging of the (NH alpha/beta)-N-15 multiplet components as a consequence of H-1 decoupling induces effective broadening of the N-15 resonance. Application of TROSY type techniques that select only the narrow component of the multiplet pattern results in an increased resolution and, thus, will be of benefit for MAS solid-state NMR Spectroscopy. (C) 2008 Wiley Periodicals, Inc
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Differential line broadening in MAS solid-state NMR due to dynamic interference.
Journal of the American Chemical Society, 2007Co-Authors: Veniamin Chevelkov, Katja Faelber, Anna Schrey, Kristina Rehbein, And Anne Diehl, Bernd ReifAbstract:Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond−microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NHα/β multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N−H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra wer...
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Differential line broadening in MAS solid-state NMR due to dynamic interference
Journal of the American Chemical Society, 2007Co-Authors: Veniamin Chevelkov, Katja Faelber, Anna Schrey, Kristina Rehbein, Anne Diehl, Bernd ReifAbstract:Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond-microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NH(alpha/beta) multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N-H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra were obtained. We expect that this approach which requires intense deuteration will have a significant impact on the quality and the rate at which solid-state NMR spectroscopic investigations will emerge in the future.
Veniamin Chevelkov - One of the best experts on this subject based on the ideXlab platform.
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TROSY effects in MAS solid-state NMR
Concepts in Magnetic Resonance Part A, 2008Co-Authors: Veniamin Chevelkov, Bernd ReifAbstract:Use of Transverse Relaxation-Optimized Spectroscopy (TROSY) type techniques had a dramatic impact on the study of large proteins with a molecular weight >30kDa for solution-state NMR. In the solid-state, such an effect would not be expected a prior, as the investigated molecules are immobilized. However, local motions induce fluctuations of the local fields experienced by the nuclear spins and, this way, are effective for relaxation. We demonstrate that protein dynamics can significantly influence the resonance line width in ultra high resolution MAS (magic angle spinning) solid-state NMR experiments. Averaging of the 15NHα/β multiplet components as a consequence of 1H decoupling induces effective broadening of the 15N resonance. Application of TROSY type techniques that select only the narrow component of the multiplet pattern results in an increased resolution and, thus, will be of benefit for MAS solid-state NMR Spectroscopy. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 143–156, 2008.
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TROSY effects in MAS solid-state NMR
Concepts in Magnetic Resonance Part A: Bridging Education and Research, 2008Co-Authors: Veniamin Chevelkov, Bernd ReifAbstract:Use of Transverse Relaxation-Optimized Spectroscopy (TROSY) type techniques had a dramatic impact on the study of large proteins with a molecular weight >30kDa for solution-state NMR. In the solid-state, such an effect would not be expected a prior, as the investigated molecules are immobilized. However, local motions induce fluctuations of the local fields experienced by the nuclear spins and, this way, are effective for relaxation. We demonstrate that protein dynamics can significantly influence the resonance line width in ultra high resolution MAS (magic angle spinning) solid-state NMR experiments. Averaging of the (NH alpha/beta)-N-15 multiplet components as a consequence of H-1 decoupling induces effective broadening of the N-15 resonance. Application of TROSY type techniques that select only the narrow component of the multiplet pattern results in an increased resolution and, thus, will be of benefit for MAS solid-state NMR Spectroscopy. (C) 2008 Wiley Periodicals, Inc
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Differential line broadening in MAS solid-state NMR due to dynamic interference.
Journal of the American Chemical Society, 2007Co-Authors: Veniamin Chevelkov, Katja Faelber, Anna Schrey, Kristina Rehbein, And Anne Diehl, Bernd ReifAbstract:Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond−microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NHα/β multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N−H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra wer...
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Differential line broadening in MAS solid-state NMR due to dynamic interference
Journal of the American Chemical Society, 2007Co-Authors: Veniamin Chevelkov, Katja Faelber, Anna Schrey, Kristina Rehbein, Anne Diehl, Bernd ReifAbstract:Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond-microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NH(alpha/beta) multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N-H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra were obtained. We expect that this approach which requires intense deuteration will have a significant impact on the quality and the rate at which solid-state NMR spectroscopic investigations will emerge in the future.
César Fernández - One of the best experts on this subject based on the ideXlab platform.
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TROSY NMR for Studies of Large Biological Macromolecules in Solution
ChemInform, 2008Co-Authors: César Fernández, Gerhard WiderAbstract:Transverse Relaxation-Optimized Spectroscopy (TROSY), in combination with isotope labeling techniques and with improvements in NMR instrumentation, have greatly extended applications of NMR Spectroscopy to large biological macromolecules that were otherwise not accessible to high-resolution solution state NMR. Important recent applications of TROSY include the structure determinations of integral membrane proteins in detergent micelles, structural and functional studies of large proteins in monomeric form and in macromolecular complexes, and investigations of intermolecular interactions in large complexes. Moreover, TROSY can improve measurements of NMR parameters, such as residual dipolar couplings and scalar couplings across hydrogen bonds, which contribute to a further improvement of the quality and the precision of solution structures of large proteins and oligonucleotides.
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Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles
Journal of Biomolecular NMR, 2003Co-Authors: Christian Hilty, Gerhard Wider, César Fernández, Kurt WüthrichAbstract:In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration. As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination. For example, 3D 15 N- and 13 C-resolved [ 1 H, 1 H]-NOESY spectra of the E. Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu. In this paper, we used biosynthetically-directed fractional 13 C labeling of OmpX and [ 13 C, 1 H]-HSQC Spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC. For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a 1 H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument. Abbreviations: 2D – two-dimensional; 3D – three-dimensional; ct – constant time; DHPC – dihexanoylphosphatidylcholine (1,2-dihexanoyl-sn-glycero-3-phosphocholine); HSQC – heteronuclear single quantum coherence; NOE – nuclear Overhauser enhancement; NOESY – NOE Spectroscopy; OmpX – outer membrane protein X from Escherichia coli; TROSY – Transverse Relaxation-Optimized Spectroscopy.
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TROSY in NMR studies of the structure and function of large biological macromolecules
Current Opinion in Structural Biology, 2003Co-Authors: César Fernández, Gerhard WiderAbstract:Abstract Transverse Relaxation-Optimized Spectroscopy (TROSY), in combination with various isotope-labeling techniques, has opened avenues to study biomolecules with molecular masses of up to 1 000 000 Da by solution NMR. Important recent applications of TROSY include the structure determination of membrane proteins in detergent micelles, structural and functional studies of large proteins in both monomeric form and macromolecular complexes, and investigations of intermolecular interactions in large complexes. TROSY improves the measurement of residual dipolar couplings and the detection of scalar couplings across hydrogen bonds — techniques that promise to further enhance the determination of solution structures of large proteins and oligonucleotides.
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Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli
FEBS Letters, 2001Co-Authors: César Fernández, Konstantin Pervushin, Christian Hilty, Koba Adeishvili, S. Bonjour, Kurt WüthrichAbstract:Abstract Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (Transverse Relaxation-Optimized Spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA β-barrel from those of OmpX. Overall, the present data demonstrate that Relaxation-Optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.
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Transverse Relaxation-Optimized NMR Spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: César Fernández, Koba Adeishvili, Kurt WüthrichAbstract:The 2H,13C,15N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse Relaxation-Optimized Spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that Relaxation-Optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.
