Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins (original) (raw)
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Biophysical journal, 2014
Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR pa...
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the 15 N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of 15 N and 2 H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.
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CURRENT OPINION IN STRUCTURAL BIOLOGY, 2011
Structure determination of membrane-associated proteins (MPs) represents a frontier of structural biology that is characterized by unique challenges in sample preparation and data acquisition. No less important is our ability to study the dynamics of MPs, since MP flexibility and characteristic motions often make sizeable contributions to their function. This review focuses on solution state NMR methods to characterize dynamics of MPs in the membrane environment. NMR approaches to study molecular motions on a wide range of timescales and their application to membrane proteins are described. Studies of polytopic and bitopic MPs demonstrating the power of such methods to characterize the dynamic behavior of MPs and their interaction with the membranemimicking surroundings are presented. Attempts are made to place the dynamic conclusions into a biological context. The importance and limitations of such investigations guarantee that further developments in this field will be actively pursued.
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Proceedings of the National Academy of Sciences, 2009
Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein...
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Conformational sampling and dynamics of membrane proteins from 10-nanosecond computer simulations
Proteins: Structure, Function, and Bioinformatics, 2004
In the current report, we provide a quantitative analysis of the convergence of the sampling of conformational space accomplished in molecular dynamics simulations of membrane proteins of duration in the order of 10 nanoseconds. A set of proteins of diverse size and topology is considered, ranging from helical pores such as gramicidin and small -barrels such as OmpT, to larger and more complex structures such as rhodopsin and FepA. Principal component analysis of the C ␣ -atom trajectories was employed to assess the convergence of the conformational sampling in both the transmembrane domains and the whole proteins, while the time-dependence of the average structure was analyzed to obtain single-domain information. The membrane-embedded regions, particularly those of small or structurally simple proteins, were found to achieve reasonable convergence. By contrast, extramembranous domains lacking secondary structure are often markedly under-sampled, exhibiting a continuous structural drift. This drift results in a significant imprecision in the calculated B-factors, which detracts from any quantitative comparison to experimental data. In view of such limitations, we suggest that similar analyses may be valuable in simulation studies of membrane protein dynamics, in order to attach a level of confidence to any biologically relevant observations. Proteins 2004;57: 783-791.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
Solid-state NMR spectroscopy is being developed at a fast pace for the structural investigation of immobilized and non-crystalline biomolecules. These include proteins and peptides associated with phospholipid bilayers. In contrast to solution NMR spectroscopy, where complete or almost complete averaging leads to isotropic values, the anisotropic character of nuclear interactions is apparent in solid-state NMR spectra. In static samples the orientation dependence of chemical shift, dipolar or quadrupolar interactions, therefore, provides angular constraints when the polypeptides have been reconstituted into oriented membranes. Furthermore, solid-state NMR spectroscopy of aligned samples offers distinct advantages in allowing access to dynamic processes such as topological equilibria or rotational diffusion in membrane environments. Alternatively, magic angle sample spinning (MAS) results in highly resolved NMR spectra, provided that the sample is sufficiently homogenous. MAS spinning solid-state NMR spectra allow to measure distances and dihedral angles with high accuracy. The technique has recently been developed to selectively establish through-space and through-bond correlations between nuclei, similar to the approaches well-established in solution-NMR spectroscopy. D
In the current report, we provide a quantitative analysis of the convergence of the sampling of conformational space accomplished in molecular dynamics simulations of membrane proteins of duration in the order of 10 nanoseconds. A set of proteins of diverse size and topology is considered, ranging from helical pores such as gramicidin and small -barrels such as OmpT, to larger and more complex structures such as rhodopsin and FepA. Principal component analysis of the C ␣-atom trajectories was employed to assess the convergence of the conformational sampling in both the transmembrane domains and the whole proteins, while the time-dependence of the average structure was analyzed to obtain single-domain information. The membrane-embedded regions, particularly those of small or structurally simple proteins, were found to achieve reasonable convergence. By contrast, extramembranous domains lacking secondary structure are often markedly under-sampled, exhibiting a continuous structural drift. This drift results in a significant imprecision in the calculated B-factors, which detracts from any quantitative comparison to experimental data. In view of such limitations, we suggest that similar analyses may be valuable in simulation studies of membrane protein dynamics, in order to attach a level of confidence to any biologically relevant observations. Proteins 2004;57: 783-791.