A solution NMR view of protein dynamics in the biological membrane (original) (raw)
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High-Resolution NMR determination of the dynamic structure of membrane proteins.
15 N spin-relaxation rates are demonstrated to provide critical information about the long-range structure and internal motions of membrane proteins. Combined with an improved calculation method, the relaxation-rate-derived structure of the 283-residue human voltage-dependent anion channel revealed an anisotropically shaped barrel with a rigidly attached N-terminal helix. Our study thus establishes an NMR spectroscopic approach to determine the structure and dynamics of mammalian membrane proteins at high accuracy and resolution.
Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides
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.
Techniques and applications of NMR to membrane proteins (Review)
Molecular Membrane Biology, 2004
The fact that membrane proteins are notoriously difficult to analyse using standard protocols for atomic-resolution structure determination methods have motivated adaptation of these techniques to membrane protein studies as well as development of new technologies. With this motivation, liquid-state nuclear magnetic resonance (NMR) has recently been used with success for studies of peptides and membrane proteins in detergent micelles, and solid-state NMR has undergone a tremendous evolution towards characterization of membrane proteins in native membrane and oriented phospholipid bilayers. In this mini-review, we describe some of the technological challenges behind these efforts and provide examples on their use in membrane biology.
Biophysical Journal, 2009
Membrane proteins and peptides exhibit a preferred orientation in the lipid bilayer while fluctuating in an anisotropic manner. Both the orientation and the dynamics have direct functional implications, but motions are usually not accessible, and structural descriptions are generally static. Using simulated data, we analyze systematically the impact of whole-body motions on the peptide orientations calculated from two-dimensional polarization inversion spin exchange at the magic angle (PISEMA) NMR. Fluctuations are found to have a significant effect on the observed spectra. Nevertheless, wheel-like patterns are still preserved, and it is possible to determine the average peptide tilt and azimuthal rotation angles using simple static models for the spectral fitting. For helical peptides undergoing large-amplitude fluctuations, as in the case of transmembrane monomers, improved fits can be achieved using an explicit dynamics model that includes Gaussian distributions of the orientational parameters. This method allows extracting the amplitudes of fluctuations of the tilt and azimuthal rotation angles. The analysis is further demonstrated by generating first a virtual PISEMA spectrum from a molecular dynamics trajectory of the model peptide, WLP23, in a lipid membrane. That way, the dynamics of the system from which the input spectrum originates is completely known at atomic detail and can thus be directly compared with the dynamic output obtained from the fit. We find that fitting our dynamics model to the polar index slant angles wheel gives an accurate description of the amplitude of underlying motions, together with the average peptide orientation.
Structure and dynamics of a membrane protein in micelles from three solution NMR experiments
2003
Three solution NMR experiments on a uniformly 15 N labeled membrane protein in micelles provide sufficient information to describe the structure, topology, and dynamics of its helices, as well as additional information that characterizes the principal features of residues in terminal and inter-helical loop regions. The backbone amide resonances are assigned with an HMQC-NOESY experiment and the backbone dynamics are characterized by a 1 H-15 N heteronuclear NOE experiment, which clearly distinguishes between the structured helical residues and the more mobile residues in the terminal and interhelical loop regions of the protein. The structure and topology of the helices are described by Dipolar waves and PISA wheels derived from experimental measurements of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). The results show that the membranebound form of Pf1 coat protein has a 20-residue trans-membrane hydrophobic helix with an orientation that differs by about 90 • from that of an 8-residue amphipathic helix. This combination of three-experiments that yields Dipolar waves and PISA wheels has the potential to contribute to high-throughput structural characterizations of membrane proteins.
Journal of the American Chemical Society, 2010
Despite recent advances in NMR approaches for structural biology, determination of membrane protein dynamics in its native environment continues to be a monumental challenge as most NMR structural studies of membrane proteins are commonly carried out in either micelles or in vesicle systems under frozen conditions. To overcome this difficulty, we propose a solid-state NMR technique that allows for the determination of side chain dynamics from membrane proteins in lipid bilayers. This new technique, namely dipolar enhanced polarization transfer (DREPT), allows for a wide range of dipolar couplings to be encoded, providing high resolution and sensitivity for systems that undergo motional averaging such as that of amino acid side chains. NMR observables such as dipolar couplings and chemical shift anisotropy, which are highly sensitive to molecular motions, provide a direct way of probing protein dynamics over a wide range of timescales. Therefore, using an appropriate model it is possible to determine side chain dynamics that can provide additional information on the topology and function of a membrane protein in its native environment. Dynamics is crucial to the function of membrane-associated molecules such as peptides and membrane proteins. 1 In particular, the dynamics of amino acid side chains play a central role in the folding and oligomerization of membrane proteins as well as in their functions including ion transport and cell membrane disruption. Therefore, an amino acid sequence encodes not only the structure of a protein but also a range of accessible dynamics, which in most cases determines its function as well as its adaptability to various stimuli such as temperature, pH and ionic strength. While solving atomic-level resolution structures of membrane-associated proteins is a challenge, characterization of their dynamics at highresolution is even more difficult. In particular, measurement of protein dynamics from physiologically-relevant fluid membranes is a major challenge as solid-state NMR structural studies of membrane proteins are commonly carried out using frozen model membranes such as multilamellar vesicles. 2-4 To overcome these limitations, we propose a 2D solid-state NMR technique that allows determination of side chain dynamics from a membrane protein in its near-native environment. This technique, namely Dipolar-Enhanced Polarization Transfer (DREPT), is a
Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR
Biology
Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics. NMR is uniquely suited to resolving atomic environments within complex molecular assemblies and reporting on membrane organisation, protein structure, lipid and polysaccharide composition, conformational variations and molecular interactions. The main challenge in membrane protein studies at the atomic level remains the need for a membrane environment to support their fold. NMR studies in membrane mimetics and membranes of increasing complexity offer close to native environments for structural and molecular studies of membrane pr...
Orientation and Dynamics of Peptides in Membranes Calculated from 2H-NMR Data
Biophysical Journal, 2009
Solid-state 2 H-NMR is routinely used to determine the alignment of membrane-bound peptides. Here we demonstrate that it can also provide a quantitative measure of the fluctuations around the distinct molecular axes. Using several dynamic models with increasing complexity, we reanalyzed published 2 H-NMR data on two representative a-helical peptides: 1), the amphiphilic antimicrobial peptide PGLa, which permeabilizes membranes by going from a monomeric surface-bound to a dimeric tilted state and finally inserting as an oligomeric pore; and 2), the hydrophobic WALP23, which is a typical transmembrane segment, although previous analysis had yielded helix tilt angles much smaller than expected from hydrophobic mismatch and molecular dynamics simulations. Their 2 H-NMR data were deconvoluted in terms of the two main helix orientation angles (representing the time-averaged peptide tilt and azimuthal rotation), as well as the amplitudes of fluctuation about the corresponding molecular axes (providing the dynamic picture). The mobility of PGLa is found to be moderate and to correlate well with the respective oligomeric states. WALP23 fluctuates more vigorously, now in better agreement with the molecular dynamics simulations and mismatch predictions. The analysis demonstrates that when 2 H-NMR data are fitted to extract peptide orientation angles, an explicit representation of the peptide rigid-body angular fluctuations should be included.