Mixing and matching detergents for membrane protein NMR structure determination (original) (raw)
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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.
F1000 - Post-publication peer review of the biomedical literature, 2010
Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity but also because the effects of slow molecular reorientation are exacerbated by the need to solublize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solublize the protein in a low viscosity solvent where the rapid tumbling of the resulting particle allows use of standard triple resonance methods. The preparation of such samples of membrane proteins is difficult. Using a 54 kDa construct of the homotetrameric potassium channel KcsA we demonstrate a strategy that employs a hybrid surfactant to transfer the protein to the reverse micelle system.
Modulating the Physical Properties of Micelles for Membrane Protein Investigations
Biophysical Journal, 2013
Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2-1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ,16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.
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.
An evaluation of detergents for NMR structural studies of membrane proteins
Journal of Biomolecular Nmr, 2004
Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D 1 H-15 N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested -R. sphaeroides LH1 α and β subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter -with 1, 2, or 4 membrane spanning α-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 β subunits form native-like complexes with bacteriochlorphyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of 15 N transverse, longitudinal and 15 N{ 1 H} nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.
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.
On choosing a detergent for solution NMR studies of membrane proteins
Journal of biomolecular NMR, 1998
Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (x10(-7) cm2/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken t...
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.
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.