Solid-state NMR spectroscopy applied to membrane proteins (original) (raw)
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Methodological development of solid-state NMR for characterization of membrane proteins
Concepts in Magnetic Resonance, 2003
Solid-state NMR is a promising method for structural analysis of membrane proteins. The method is capable of providing atomic-resolution structure information without the need for crystals or fast molecular motion as is required for X-ray diffraction and liquid-state NMR, respectively. However, it is clear that solid-state NMR still needs substantial methodological development before it can provide the desired detailed information about large structures or be applied routinely for structure-function relationship studies. Here, we address some important considerations and elements in the solid-state NMR methodology relevant for characterization of membrane proteins. These are relevant for establishing the appropriate technology and may serve as an inspiration for future developments in synthesis, isotope labeling, and physical preparation of samples for solid-state NMR characterization
Concepts and Methods of Solid-State NMR Spectroscopy Applied to Biomembranes
Concepts of solid-state NMR spectroscopy and applications to fluid membranes are reviewed in this paper. Membrane lipids with 2 H-labeled acyl chains or polar head groups are studied using 2 H NMR to yield knowledge of their atomistic structures in relation to equilibrium properties. This review demonstrates the principles and applications of solid-state NMR by unifying dipolar and quadrupolar interactions and highlights the unique features offered by solid-state 2 H NMR with experimental illustrations. For randomly oriented multilamellar lipids or aligned membranes, solid-state 2 H NMR enables direct measurement of residual quadrupolar couplings (RQCs) due to individual C− 2 H-labeled segments. The distribution of RQC values gives nearly complete profiles of the segmental order parameters S CD (i) as a function of acyl segment position (i). Alternatively, one can measure residual dipolar couplings (RDCs) for natural abundance lipid samples to obtain segmental S CH order parameters. A theoretical mean-torque model provides acyl-packing profiles representing the cumulative chain extension along the normal to the aqueous interface. Equilibrium structural properties of fluid bilayers and various thermodynamic quantities can then be calculated, which describe the interactions with cholesterol, detergents, peptides, and integral membrane proteins and formation of lipid rafts. One can also obtain direct information for membrane-bound peptides or proteins by measuring RDCs using magic-angle spinning (MAS) in combination with dipolar recoupling methods. Solid-state NMR methods have been extensively applied to characterize model membranes and membrane-bound peptides and proteins, giving unique information on their conformations, orientations, and interactions in the natural liquid-crystalline state. CONTENTS
Solid-state NMR structures of integral membrane proteins
Molecular Membrane Biology, 2015
Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid-and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.
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.
Sensitivity and resolution enhancement of oriented solid-state NMR: Application to membrane proteins
Progress in Nuclear Magnetic Resonance Spectroscopy, 2013
Oriented solid-state NMR (O-ssNMR) spectroscopy is a major technique for the high-resolution analysis of the structure and topology of transmembrane proteins in native-like environments. Unlike magic angle spinning (MAS) techniques, O-ssNMR spectroscopy requires membrane protein preparations that are uniformly oriented (mechanically or magnetically) so that anisotropic NMR parameters, such as dipolar and chemical shift interactions, can be measured to determine structure and orientation of membrane proteins in lipid bilayers. Traditional sample preparations involving mechanically aligned lipids often result in short relaxation times which broaden the 15 N resonances and encumber the manipulation of nuclear spin coherences. The introduction of lipid bicelles as membrane mimicking systems has changed this scenario, and the more favorable relaxation properties of membrane protein 15 N and 13 C resonances make it possible to develop new, more elaborate pulse sequences for higher spectral resolution and sensitivity. Here, we describe our recent progress in the optimization of O-ssNMR pulse sequences. We explain the theory behind these experiments, demonstrate their application to small and medium size proteins, and describe the technical details for setting up these new experiments on the new generation of NMR spectrometers.
Solid-state NMR Structure Determination
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2003
Biological applications of solid-state NMR (SS-NMR) have been predominantly in the area of model membrane systems. Increasingly the focus has been membrane peptides and proteins. SS-NMR is able to provide information about how the peptides or proteins interact with the lipids or other peptides/proteins in the membrane, their effect on the membrane and the location of the peptides or proteins relative to the membrane surface. Recent developments in biological SS-NMR have been made possible by improvements in labelling and NMR techniques. This review discusses aligned systems and magic angle spinning techniques, bilayers and bicelles, and measurement of chemical shift anisotropy and dipolar coupling. A number of specific experiments such as cross polarization, rotational resonance, REDOR, PISEMA, MAOSS and multidimensional experiments are described. In addition to 2 H, 13 C and 15 N, recent solid-sate 1 H, 19 F and 17 O NMR work is discussed. Several examples of the use of SS-NMR to determine the structure of membrane peptides and proteins are given. IUBMB Life, 55: 515-523, 2003
Journal of magnetic resonance (San Diego, Calif. : 1997), 2017
Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is emerging as a unique method for the atomic resolution structure determination of native membrane proteins in lipid bilayers. AlthoughC-detected ssNMR experiments continue to play a major role, recent technological developments have made it possible to carry outH-detected experiments, boosting both sensitivity and resolution. Here, we describe a new set ofH-detected hybrid pulse sequences that combine through-bond and through-space correlation elements into single experiments, enabling the simultaneous detection of rigid and dynamic domains of membrane proteins. As proof-of-principle, we applied these new pulse sequences to the membrane protein phospholamban (PLN) reconstituted in lipid bilayers under moderate MAS conditions. The cross-polarization (CP) based elements enabled the detection of the relatively immobile residues of PLN in the transmembrane domain using through-space correlations; whereas the most dynamic ...
Methods, 2018
The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions. Here, we review our recent efforts to develop single and dual acquisition pulse sequences with either 1 H or 13 C detection that utilize both DC and J-coupling mediated transfer to detect both rigid and mobile regions of membrane proteins in native-like lipid environments. We show the application of these new methods for studying the conformational equilibrium of a single-pass membrane protein, phospholamban, which regulates the calcium transport across the sarcoplasmic reticulum (SR) membrane by interacting with the SR Ca 2+-ATPase. We anticipate that these methods will be ideal to portray the complex dynamics of membrane proteins in their native environments.