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G. Wagner - One of the best experts on this subject based on the ideXlab platform.
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5.7 Solution NMR Spectroscopy of Integral Membrane Proteins
Comprehensive Biophysics, 2020Co-Authors: S. Hiller, G. WagnerAbstract:Solution nuclear magnetic resonance (NMR) spectroscopy is a generally applicable method for studying structure and function of integral membrane proteins at atomic reSolution. It provides unique features complementary to other high-reSolution techniques. This chapter presents an overview of current Solution NMR techniques for integral membrane proteins, describes their potentials and limitations, and reviews successful structure determinations as well as selected functional studies. The discussion includes several practical examples from Solution NMR studies of the human voltage-dependent anion channel.
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VDAC Studied by Solution NMR: Implications for the Native Structure
Biophysical Journal, 2020Co-Authors: S. Hiller, Tsyr-yan Yu, Thomas Raschle, Amanda J. Rice, Thomas Walz, G. WagnerAbstract:The voltage-dependent anion channel (VDAC) is the main pathway for metabolites, small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC has been extensively studied for over thirty years and recently, high-reSolution structures of VDAC were determined by X-ray and NMR methods (1-3). These studies used recombinant, refolded protein in membrane mimicking environments and thus the valid question arises how well the resulting atomic structure might resemble the “native” structure of VDAC in the mitochondrial outer membrane (4).Here, we describe implications from Solution NMR experiments to this question. Recombinant human VDAC-1 is stably folded in LDAO detergent micelles. Well resolved NMR spectra, including four-dimensional NOESYs, yielded a consistent set of more than 1000 spatial spin-spin correlations that unambiguously define the three-dimensional structure of VDAC-1 (1). The protein forms a 19-stranded beta-barrel with 18 antiparallel and 1 parallel strand pairing. The N-terminal 25 residues are not part of the beta-barrel and Solution NMR data link the dynamic properties of this segment to the well-known voltage gating process. The inner diameter of the VDAC-1 barrel is about 25 A, in consistence with published micrographs of native or native-like preparations. The entire outside perimeter of the barrel is hydrophobic and covered by detergent molecules, compatible with a membrane bilayer topology. NMR measurements also revealed interactions of VDAC-1 with beta-NADH and cholesterol, providing a functional connection to experiments on native states of the protein. Furthermore, we can link the micelle-bound state of VDAC structurally and functionally to preparations in phospholipid bilayers by comparing NMR spectra and electron micrographs.(1) Hiller et al. Science 321, 1206 (2008).(2) Bayrhuber et al. PNAS 105, 15370 (2008).(3) Ujwal et al. PNAS 105, 17742 (2008).(4) Colombini. Trends Biochem. Sci. 34, 382 (2009).
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emerging Solution NMR methods to illuminate the structural and dynamic properties of proteins
Current Opinion in Structural Biology, 2019Co-Authors: Haribabu Arthanari, Koh Takeuchi, Abhinav Dubey, G. WagnerAbstract:The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-reSolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Here, we summarize recent technical landmarks in Solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic reSolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.
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nonmicellar systems for Solution NMR spectroscopy of membrane proteins
Current Opinion in Structural Biology, 2010Co-Authors: Thomas Raschle, S. Hiller, Manuel Etzkorn, G. WagnerAbstract:Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, nonmicellar solubilization techniques, with a particular focus on their application to Solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.
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the role of Solution NMR in the structure determinations of vdac 1 and other membrane proteins
Current Opinion in Structural Biology, 2009Co-Authors: S. Hiller, G. WagnerAbstract:The voltage-dependent anion channel (VDAC) is an essential protein in the eukaryotic outer mitochondrial membrane, providing the pore for substrate diffusion. Three high-reSolution structures of the isoform 1 of VDAC in detergent micelles and bicelles have recently been published, using Solution NMR and X-ray crystallography. They resolve longstanding discussions about the membrane topology of VDAC and provide the first eukaryotic β-barrel membrane protein structure. The structure contains a surprising feature that had not been observed in an integral membrane protein before: A parallel β-strand pairing and thus an odd number of strands. The studies also give a structural and functional basis for the voltage gating mechanism of VDAC and its modulation by NADH; however, they do not fully explain these functions yet. With the de novo structure of VDAC-1, as well as those of half a dozen other proteins, the number of integral membrane protein structures solved by Solution NMR has doubled in the past two years. Numerous further structural and functional studies on many different membrane proteins show that Solution NMR has become an important tool for membrane protein molecular biology.
Koh Takeuchi - One of the best experts on this subject based on the ideXlab platform.
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spotlight on the ballet of proteins the structural dynamic properties of proteins illuminated by Solution NMR
International Journal of Molecular Sciences, 2020Co-Authors: Yuji Tokunaga, Thibault Viennet, Haribabu Arthanari, Koh TakeuchiAbstract:Solution NMR spectroscopy is a unique and powerful technique that has the ability to directly connect the structural dynamics of proteins in physiological conditions to their activity and function. Here, we summarize recent studies in which Solution NMR contributed to the discovery of relationships between key dynamic properties of proteins and functional mechanisms in important biological systems. The capacity of NMR to quantify the dynamics of proteins over a range of time scales and to detect lowly populated protein conformations plays a critical role in its power to unveil functional protein dynamics. This analysis of dynamics is not only important for the understanding of biological function, but also in the design of specific ligands for pharmacologically important proteins. Thus, the dynamic view of structure provided by NMR is of importance in both basic and applied biology.
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structure determination using Solution NMR is it worth the effort
Journal of Magnetic Resonance, 2019Co-Authors: Koh Takeuchi, Kumaran Baskaran, Haribabu ArthanariAbstract:Abstract It has been almost 40 years since Solution NMR joined X-ray crystallography as a technique for determining high-reSolution structures of proteins. Since then NMR derived structure has contributed in fundamental ways to our understanding of the function of biomolecules. With the already existing mature field of X-ray crystallography and the emergence of cryo-EM as techniques to tackle high-reSolution structures of large protein complexes, the role of NMR in structure determination has been questioned. However, NMR has the unique ability to recapitulate the dynamic motion of proteins in their structures, while size limitations of the biomolecular systems that can be routinely studied still present challenges. The field has continually developed methodology and instrumentation since its introduction, pushing its frontiers and redefining its limits. Here we present a brief overview of NMR-based structure determination over the past 40 years. We outline the current state of the field and look ahead to the challenges that still need to be addressed to realize the future potential of NMR as a structural technique.
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emerging Solution NMR methods to illuminate the structural and dynamic properties of proteins
Current Opinion in Structural Biology, 2019Co-Authors: Haribabu Arthanari, Koh Takeuchi, Abhinav Dubey, G. WagnerAbstract:The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-reSolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Here, we summarize recent technical landmarks in Solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic reSolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.
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Functional dynamics of proteins revealed by Solution NMR.
Current Opinion in Structural Biology, 2012Co-Authors: Masanori Osawa, Koh Takeuchi, Takumi Ueda, Noritaka Nishida, Ichio ShimadaAbstract:Solution NMR spectroscopy can analyze the dynamics of proteins on a wide range of timescales, from picoseconds to even days, in a site-specific manner, and thus its results are complementary to the detailed but largely static structural information obtained by X-ray crystallography. We review recent progresses in a variety of NMR techniques, including relaxation dispersion and paramagnetic relaxation enhancement (PRE), that permit the observation of the low-populated states, which had been ‘invisible’ with other techniques. In addition, we review how NMR spectroscopy can be used to elucidate functionally relevant protein dynamics.
Haribabu Arthanari - One of the best experts on this subject based on the ideXlab platform.
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spotlight on the ballet of proteins the structural dynamic properties of proteins illuminated by Solution NMR
International Journal of Molecular Sciences, 2020Co-Authors: Yuji Tokunaga, Thibault Viennet, Haribabu Arthanari, Koh TakeuchiAbstract:Solution NMR spectroscopy is a unique and powerful technique that has the ability to directly connect the structural dynamics of proteins in physiological conditions to their activity and function. Here, we summarize recent studies in which Solution NMR contributed to the discovery of relationships between key dynamic properties of proteins and functional mechanisms in important biological systems. The capacity of NMR to quantify the dynamics of proteins over a range of time scales and to detect lowly populated protein conformations plays a critical role in its power to unveil functional protein dynamics. This analysis of dynamics is not only important for the understanding of biological function, but also in the design of specific ligands for pharmacologically important proteins. Thus, the dynamic view of structure provided by NMR is of importance in both basic and applied biology.
-
structure determination using Solution NMR is it worth the effort
Journal of Magnetic Resonance, 2019Co-Authors: Koh Takeuchi, Kumaran Baskaran, Haribabu ArthanariAbstract:Abstract It has been almost 40 years since Solution NMR joined X-ray crystallography as a technique for determining high-reSolution structures of proteins. Since then NMR derived structure has contributed in fundamental ways to our understanding of the function of biomolecules. With the already existing mature field of X-ray crystallography and the emergence of cryo-EM as techniques to tackle high-reSolution structures of large protein complexes, the role of NMR in structure determination has been questioned. However, NMR has the unique ability to recapitulate the dynamic motion of proteins in their structures, while size limitations of the biomolecular systems that can be routinely studied still present challenges. The field has continually developed methodology and instrumentation since its introduction, pushing its frontiers and redefining its limits. Here we present a brief overview of NMR-based structure determination over the past 40 years. We outline the current state of the field and look ahead to the challenges that still need to be addressed to realize the future potential of NMR as a structural technique.
-
emerging Solution NMR methods to illuminate the structural and dynamic properties of proteins
Current Opinion in Structural Biology, 2019Co-Authors: Haribabu Arthanari, Koh Takeuchi, Abhinav Dubey, G. WagnerAbstract:The first recognition of protein breathing was more than 50 years ago. Today, we are able to detect the multitude of interaction modes, structural polymorphisms, and binding-induced changes in protein structure that direct function. Solution-state NMR spectroscopy has proved to be a powerful technique, not only to obtain high-reSolution structures of proteins, but also to provide unique insights into the functional dynamics of proteins. Here, we summarize recent technical landmarks in Solution NMR that have enabled characterization of key biological macromolecular systems. These methods have been fundamental to atomic reSolution structure determination and quantitative analysis of dynamics over a wide range of time scales by NMR. The ability of NMR to detect lowly populated protein conformations and transiently formed complexes plays a critical role in its ability to elucidate functionally important structural features of proteins and their dynamics.
S. Hiller - One of the best experts on this subject based on the ideXlab platform.
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5.7 Solution NMR Spectroscopy of Integral Membrane Proteins
Comprehensive Biophysics, 2020Co-Authors: S. Hiller, G. WagnerAbstract:Solution nuclear magnetic resonance (NMR) spectroscopy is a generally applicable method for studying structure and function of integral membrane proteins at atomic reSolution. It provides unique features complementary to other high-reSolution techniques. This chapter presents an overview of current Solution NMR techniques for integral membrane proteins, describes their potentials and limitations, and reviews successful structure determinations as well as selected functional studies. The discussion includes several practical examples from Solution NMR studies of the human voltage-dependent anion channel.
-
VDAC Studied by Solution NMR: Implications for the Native Structure
Biophysical Journal, 2020Co-Authors: S. Hiller, Tsyr-yan Yu, Thomas Raschle, Amanda J. Rice, Thomas Walz, G. WagnerAbstract:The voltage-dependent anion channel (VDAC) is the main pathway for metabolites, small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC has been extensively studied for over thirty years and recently, high-reSolution structures of VDAC were determined by X-ray and NMR methods (1-3). These studies used recombinant, refolded protein in membrane mimicking environments and thus the valid question arises how well the resulting atomic structure might resemble the “native” structure of VDAC in the mitochondrial outer membrane (4).Here, we describe implications from Solution NMR experiments to this question. Recombinant human VDAC-1 is stably folded in LDAO detergent micelles. Well resolved NMR spectra, including four-dimensional NOESYs, yielded a consistent set of more than 1000 spatial spin-spin correlations that unambiguously define the three-dimensional structure of VDAC-1 (1). The protein forms a 19-stranded beta-barrel with 18 antiparallel and 1 parallel strand pairing. The N-terminal 25 residues are not part of the beta-barrel and Solution NMR data link the dynamic properties of this segment to the well-known voltage gating process. The inner diameter of the VDAC-1 barrel is about 25 A, in consistence with published micrographs of native or native-like preparations. The entire outside perimeter of the barrel is hydrophobic and covered by detergent molecules, compatible with a membrane bilayer topology. NMR measurements also revealed interactions of VDAC-1 with beta-NADH and cholesterol, providing a functional connection to experiments on native states of the protein. Furthermore, we can link the micelle-bound state of VDAC structurally and functionally to preparations in phospholipid bilayers by comparing NMR spectra and electron micrographs.(1) Hiller et al. Science 321, 1206 (2008).(2) Bayrhuber et al. PNAS 105, 15370 (2008).(3) Ujwal et al. PNAS 105, 17742 (2008).(4) Colombini. Trends Biochem. Sci. 34, 382 (2009).
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Characterization of the insertase BamA in three different membrane mimetics by Solution NMR spectroscopy.
Journal of biomolecular NMR, 2015Co-Authors: Leonor Morgado, Kornelius Zeth, Björn M Burmann, Timm Maier, S. HillerAbstract:The insertase BamA is the central protein of the Bam complex responsible for outer membrane protein biogenesis in Gram-negative bacteria. BamA features a 16-stranded transmembrane β-barrel and five periplasmic POTRA domains, with a total molecular weight of 88 kDa. Whereas the structure of BamA has recently been determined by X-ray crystallography, its functional mechanism is not well understood. This mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and NMR spectroscopy is thus a method of choice for its elucidation. Here, we report Solution NMR studies of different BamA constructs in three different membrane mimetic systems: LDAO micelles, DMPC:DiC7PC bicelles and MSP1D1:DMPC nanodiscs. The impact of biochemical parameters on the spectral quality was investigated, including the total protein concentration and the detergent:protein ratio. The barrel of BamA is folded in micelles, bicelles and nanodiscs, but the N-terminal POTRA5 domain is flexibly unfolded in the absence of POTRA4. Measurements of backbone dynamics show that the variable insertion region of BamA, located in the extracellular lid loop L6, features high local flexibility. Our work establishes biochemical preparation schemes for BamA, which will serve as a platform for structural and functional studies of BamA and its role within the Bam complex by Solution NMR spectroscopy.
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nonmicellar systems for Solution NMR spectroscopy of membrane proteins
Current Opinion in Structural Biology, 2010Co-Authors: Thomas Raschle, S. Hiller, Manuel Etzkorn, G. WagnerAbstract:Integral membrane proteins play essential roles in many biological processes, such as energy transduction, transport of molecules, and signaling. The correct function of membrane proteins is likely to depend strongly on the chemical and physical properties of the membrane. However, membrane proteins are not accessible to many biophysical methods in their native cellular membrane. A major limitation for their functional and structural characterization is thus the requirement for an artificial environment that mimics the native membrane to preserve the integrity and stability of the membrane protein. Most commonly employed are detergent micelles, which can however be detrimental to membrane protein activity and stability. Here, we review recent developments for alternative, nonmicellar solubilization techniques, with a particular focus on their application to Solution NMR studies. We discuss the use of amphipols and lipid bilayer systems, such as bicelles and nanolipoprotein particles (NLPs). The latter show great promise for structural studies in near native membranes.
-
the role of Solution NMR in the structure determinations of vdac 1 and other membrane proteins
Current Opinion in Structural Biology, 2009Co-Authors: S. Hiller, G. WagnerAbstract:The voltage-dependent anion channel (VDAC) is an essential protein in the eukaryotic outer mitochondrial membrane, providing the pore for substrate diffusion. Three high-reSolution structures of the isoform 1 of VDAC in detergent micelles and bicelles have recently been published, using Solution NMR and X-ray crystallography. They resolve longstanding discussions about the membrane topology of VDAC and provide the first eukaryotic β-barrel membrane protein structure. The structure contains a surprising feature that had not been observed in an integral membrane protein before: A parallel β-strand pairing and thus an odd number of strands. The studies also give a structural and functional basis for the voltage gating mechanism of VDAC and its modulation by NADH; however, they do not fully explain these functions yet. With the de novo structure of VDAC-1, as well as those of half a dozen other proteins, the number of integral membrane protein structures solved by Solution NMR has doubled in the past two years. Numerous further structural and functional studies on many different membrane proteins show that Solution NMR has become an important tool for membrane protein molecular biology.
Kathleen G Valentine - One of the best experts on this subject based on the ideXlab platform.
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Characterizing Protein Hydration Dynamics Using Solution NMR Spectroscopy.
Methods in Enzymology, 2018Co-Authors: Christine Jorge, Kathleen G Valentine, Bryan S. Marques, A. Joshua WandAbstract:Abstract Protein hydration is a critical aspect of protein stability, folding, and function and yet remains difficult to characterize experimentally. Solution NMR offers a route to a site-resolved view of the dynamics of protein–water interactions through the nuclear Overhauser effects between hydration water and the protein in the laboratory (NOE) and rotating (ROE) frames of reference. However, several artifacts and limitations including contaminating contributions from bulk water potentially plague this general approach and the corruption of measured NOEs and ROEs by hydrogen exchange-relayed magnetization. Fortunately, encapsulation of single protein molecules within the water core of a reverse micelle overcomes these limitations. The main advantages are the suppression hydrogen exchange and elimination of bulk water. Here we detail guidelines for the preparation Solutions of encapsulated proteins that are suitable for characterization by NOE and ROE spectroscopy. Emphasis is placed on understanding the contribution of detected NOE intensity arising from magnetization relayed by hydrogen exchange. Various aspects of fitting obtained NOE, selectively decoupled NOE, and ROE time courses are illustrated.
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reverse micelles as a platform for dynamic nuclear polarization in Solution NMR of proteins
Journal of the American Chemical Society, 2014Co-Authors: Kathleen G Valentine, Nathaniel V. Nucci, Guinevere Mathies, Sabrina Bedard, Igor Dodevski, Matthew A Stetz, Robert G Griffin, Joshua A WandAbstract:Despite tremendous advances in recent years, Solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk Solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of Solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼−93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of Solution NMR spectroscopy of proteins and other biomolecules.
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A Method for Solution NMR Structural Studies of Large Integral Membrane Proteins: Reverse Micelle Encapsulation
Biochimica et Biophysica Acta, 2009Co-Authors: Joseph M. Kielec, Kathleen G Valentine, A. Joshua WandAbstract:The structural study of membrane proteins perhaps represents one of the greatest challenges of the post-genomic era. While membrane proteins comprise over 50% of current and potential drug targets, their structural characterization lags far behind that of soluble proteins. Nuclear magnetic resonance (NMR) offers great potential not only with respect to structural characterization of integral membrane proteins but may also provide the ability to study the details of small ligand interactions. However, the size limitations of Solution NMR have restricted comprehensive structural characterization of membrane protein NMR structures to the relatively small β-barrel proteins or helical proteins of relatively simple topology. In an effort to escape the barriers presented by slow molecular reorientation of large integral membrane proteins solubilized by detergent micelles in water, we have adapted the reverse micelle encapsulation strategy originally developed for the study of large soluble proteins by Solution NMR methods. Here we review a novel approach to the solubilization of large integral membrane proteins in reverse micelle surfactants dissolved in low viscosity alkane solvents. The procedure is illustrated with a 54 kDa construct of the homotetrameric KcsA potassium channel.
