Faculty of 1000 evaluation for Reverse micelles in integral membrane protein structural biology by solution NMR spectroscopy (original) (raw)

Reverse Micelle Encapsulation of Membrane-Anchored Proteins for Solution NMR Studies

Structure, 2010

Perhaps 5 to 10% of proteins bind to the membranes via a covalently attached lipid. Post-translational attachment of fatty acids such as myristate occurs on a variety of viral and cellular proteins. Highresolution information about the nature of lipidated proteins is remarkably sparse, often because of solubility problems caused by the exposed fatty acids. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the Ca +2 signaling pathway in vision and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol(4,5) bisphosphate. Both proteins have been successfully encapsulated in the lipid extruded state and highresolution NMR spectra obtained. Both proteins bind their activating ligands in the reverse micelle. This approach seems broadly applicable to membrane proteins with exposed fatty acid chains that have eluded structural characterization by conventional approaches.

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.

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Protein Science, 2006

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of crossrelaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with ,180 SDS molecules per channel. K + ions bind to the micelle-solubilized channel with a K D of 3 6 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel. ; fax: (301) 402-0907.

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Methods, 2007

Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na, K-ATPase.

Mixing and matching detergents for membrane protein NMR structure determination

Journal of the …, 2009

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, and small angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2011

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.

Nonmicellar systems for solution NMR spectroscopy of membrane proteins

Current Opinion in Structural Biology, 2010

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, non-micellar 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.

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.

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.