Membrane Protein Structural Biology Research Articles

Lipidic phase membrane protein serial femtosecond crystallography

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

Memprotmd: Adding the Grease to Membrane Protein Structures

Membrane protein structural biology is one of the key biochemical challenges. With continuous improvements to the methods used by structural biologists there is a predicted exponential growth in the number of membrane proteins structures. Nevertheless, these biological assemblies are usually resolved in the absence of the native lipid environment. Coarse-Grained molecular dynamics (CGMD) simulations provide a means for assessing the assembly and interactions of molecular complexes at a reduced level of representation. This method has been shown to accurately predict the position and orientation of proteins within a cell membrane. The results of these predictions are available in a database (http://sbcb.bioch.ox.ac.uk/cgdb). We are in the process of pipelining the procedure, so that new membrane protein structures are automatically inserted into a DPPC lipid bilayer on release from the Protein Data Bank (PDB). The CG simulations are then assessed for protein-lipid interactions, bilayer deformation, lipid diffusion and protein tilt. The resulting models are then refined to include more physiologically relevant lipid mixtures and subsequently converted to an atomistic resolution [1] to enable more detailed simulations of lipid protein interactions [2].[1] Stansfeld, JCTC, 7 (2011) 1157-1166.[2] Stansfeld, Biochemistry, 48 (2009) 10926-33.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Neurotoxin II Bound to Acetylcholine Receptors in Native Membranes Studied by Dynamic Nuclear Polarization NMR

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.

Tolerance to Changes in Membrane Lipid Composition as a Selected Trait of Membrane Proteins

Membrane lipid composition can vary dramatically across the three domains of life and even within single organisms. Here we review evidence that the lipid-exposed surfaces of membrane proteins have generally evolved to maintain correct structure and function in the face of major changes in lipid composition. Such tolerance has allowed evolution to extensively remodel membrane lipid compositions during the emergence of new species without having to extensively remodel the associated membrane proteins. The tolerance of membrane proteins also permits single-cell organisms to vary their membrane lipid composition in response to their changing environments and allows dynamic and organelle-specific variations in the lipid compositions of eukaryotic cells. Membrane protein structural biology has greatly benefited from this seemingly intrinsic property of membrane proteins: the majority of structures determined to date have been characterized under model membrane conditions that little resemble those of native membranes. Nevertheless, with a few notable exceptions, most experimentally determined membrane protein structures appear, to a good approximation, to faithfully report on native structure.

Multiscale Simulations of Lipid Interactions with Integral Membrane Proteins: Aquaporins

Membrane protein structural biology is one of the key biochemical challenges of the coming decade. With continuous improvements to the methods used by structural biologists there is a predicted exponential growth in the number of membrane proteins structures. Nevertheless, these biological assemblies are usually resolved in the absence of the native lipid environment. Coarse-Grained molecular dynamics (CGMD) simulations provide a means for assessing the assembly and interactions of molecular complexes at a reduced level of representation. This method has been shown to accurately predict the insertion position of proteins within a cell membrane. Previous studies enabled us to correlate predicted contacts to lipids with experimental data on lipid-exposed residues for a number of membrane proteins (e.g. LacY, rhodopsin, KcsA, MscL, FepA, BtuB). The recent determination of high resolution structures for aquaporins in a phospholipid bilayer environment (Aqp0: 2B6O and 3M9I; Aqp4: 2ZZ9) has provided an excellent test case. The CGMD approach has been used to simulate lipid/protein interactions for >30 members of the aquaporin family, revealing that patterns of protein/lipid headgroup interactions are conserved and are in good agreement with the lipids resolved in the electron crystallography structures. We have extended this further by using a multi-scale approach, investigating the interactions at atomic resolution. At this scale we may also consider hydrogen bonding interactions, which also show good agreement with the experimental structures. This methodology is being incorporated into a semi-automated high-throughput pipeline to enrich our database of CGMD membrane protein simulations (CGDB; http://sbcb.bioch.ox.ac.uk/cgdb).

RAMP-ing up Class-B GPCR ECD Structural Coverage

The cocrystal structure of the heterodimer GPCR extracellular domain complex of the CGRP receptor (CLR and RAMP1) has been determined in an apo form and with two different antagonists olcegepant and telcagepant (ter Haar et al., 2010).

The high-throughtput membrane protein structural biology pipeline at the SGC

The high-throughtput membrane protein structural biology pipeline at the SGC

Deletion of a terminal residue disrupts oligomerization of a transmembrane α-helixThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

In studies of the structural biology of membrane proteins, the success of strategies based on the "divide and conquer" approach, where peptides are used to model the individual transmembrane (TM) alpha-helices of membrane proteins, depends on the correct identification of the membrane-embedded TM alpha-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of a Lys-tagged TM2 alpha-helix of myelin proteolipid protein (PLP), of parent sequence KKKK-66AFQYVIYGTASFFFLYGALLLAEG89-KKKK along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and of a hydrophobic mutant Leu-90. Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS), we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is enough to change its intrinsic properties, with TM2 66-89 displaying only a monomeric form, but with dimers arising for the other 3 peptides. A novel use of trifluoroethanol (TFE) as a maximal helix-supporting solvent demonstrated that peptides containing residues at positions 90 and (or) 90-91 displayed significantly increased helical content vs. the TM2 parent peptide. The findings suggest that deletion of critical C-terminal residue(s) tends to reposition the helix terminus toward the membrane-aqueous interface. Our overall results emphasize the potential influence of boundary residues on TM properties when using peptides as models for TM alpha-helices, and may implicate a role for these residues in membrane protein folding and assembly.

Proton-Evolved Local-Field Solid-State NMR Studies of Cytochrome b5 Embedded in Bicelles, Revealing both Structural and Dynamical Information

Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented two-dimensional proton-evolved local-field (2D PELF) pulse sequences using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically aligned bicelles containing uniformly (15)N-labeled cytochrome b(5) are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF-based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.

Oligomerization of Transmembrane Alpha-Helices Modulated By C-terminal Boundary Residues

In studies of the structural biology of membrane proteins, the success of strategies based on the “divide-and-conquer” approach, where peptides are used to model the individual transmembrane (TM) α-helices of membrane proteins, depends upon the correct identification of the membrane-embedded TM α-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of the TM2 α-helix of myelin proteolipid protein (PLP). Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS) to study parent sequence KKKK-61AFQYVIYGTASFFFLYGALL-LAEG89-KKKK - along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and a hydrophobic mutant Leu-90 - we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is sufficient to change its intrinsic properties, with TM2 61-89 displaying only a monomeric form, but with principally dimers arising for the other three peptides. The findings suggest that deletion of critical C-terminal residue(s) tends to re-position the helix terminus toward the membrane-aqueous interface, and emphasize the potential influence of boundary residues on TM properties when utilizing peptides as models for TM α-helices. These finding may implicate a role for such residues in membrane protein folding and assembly.

SDS Micelles as a Membrane-Mimetic Environment for Transmembrane Segments

An inherent dilemma in the study of the structural biology of membrane proteins is that it is often necessary to use detergents to mimic the native lipid bilayer environment. This situation is of particular interest because the generation of high-resolution structures (through X-ray crystallography and solution NMR) has overwhelmingly relied upon identification of detergents in which membrane proteins may be solubilized without denaturation into a nonbiological state. While sodium dodecyl sulfate (SDS) is perhaps the most widely employed micelle-forming detergent for laboratory procedures involving membrane proteins, it has generally been regarded as a "harsh" detergent synonymous with membrane protein denaturation. Here we investigate systematically the SDS-solubilized states of a series of model alpha-helical transmembrane (TM) segments of varying Ala and Ile content in conjunction with selected single-Asn polar substitutions. Using Lys-tagged peptides typified by KKKKK-FAIAIAIIAWAIAIIAIAIAI-KKKKK in a series of circular dichroism, fluorescence, TOXCAT dimerization assay, and SDS-PAGE migration experiments, we find that both the local environment of the individual peptide helical surfaces and the formation of oligomeric states within the SDS-peptide complex are highly sensitive to point changes in peptide sequence, particularly with respect to local segment hydrophobicity and polar residue position. The overall results suggest that detergent micelles formed from SDS are largely capable of mimicking the tertiary interactions of protein-, lipid-, and aqueous-exposed helical surfaces that arise in the folded TM domains of proteins. The molecular characteristics of SDS-peptide complexes may thus portend a corresponding role for similar TM sequences in the in vivo assembly of polytopic membrane proteins.

Efforts for structural biology of membrane proteins in a SESAME member country

Efforts for structural biology of membrane proteins in a SESAME member country

Reverse micelles in integral membrane protein structural biology by solution NMR spectroscopy.

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 solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solubilize the protein in a low-viscosity solvent in which the rapid tumbling of the resulting particle allows for 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.

Over-Expression, Solubilization, and Purification of G Protein-Coupled Receptors for Structural Biology

With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques.

NMR of Membrane-Associated Peptides and Proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.

Molecular dynamics simulations and membrane protein structure quality

Despite a growing repertoire of membrane protein structures (currently approximately 120 unique structures), considerations of low resolution and crystallization in the absence of a lipid bilayer require the development of techniques to assess the global quality of membrane protein folds. This is also the case for assessment of, e.g. homology models of human membrane proteins based on structures of (distant) bacterial homologues. Molecular dynamics (MD) simulations may be used to help evaluate the quality of a membrane protein structure or model. We have used a structure of the bacterial ABC transporter MsbA which has the correct transmembrane helices but an incorrect handedness and topology of their packing to test simulation methods of quality assessment. An MD simulation of the MsbA model in a lipid bilayer is compared to a simulation of another bacterial ABC transporter, BtuCD. The latter structure has demonstrated good conformational stability in the same bilayer environment and over the same timescale (20 ns) as for the MsbA model simulation. A number of comparative analyses of the two simulations were performed to assess changes in the structural integrity of each protein. The results show a significant difference between the two simulations, chiefly due to the dramatic structural deformations of MsbA. We therefore propose that MD could become a useful quality control tool for membrane protein structural biology. In particular, it provides a way in which to explore the global conformational stability of a model membrane protein fold.

How Does a Voltage Sensor Interact with a Lipid Bilayer? Simulations of a Potassium Channel Domain

SummaryThe nature of voltage sensing by voltage-activated ion channels is a key problem in membrane protein structural biology. The way in which the voltage-sensor (VS) domain interacts with its membrane environment remains unclear. In particular, the known structures of Kv channels do not readily explain how a positively charged S4 helix is able to stably span a lipid bilayer. Extended (2 × 50 ns) molecular dynamics simulations of the high-resolution structure of the isolated VS domain from the archaebacterial potassium channel KvAP, embedded in zwitterionic and in anionic lipid bilayers, have been used to explore VS/lipid interactions at atomic resolution. The simulations reveal penetration of water into the center of the VS and bilayer. Furthermore, there is significant local deformation of the lipid bilayer by interactions between lipid phosphate groups and arginine side chains of S4. As a consequence of this, the electrostatic field is “focused” across the center of the bilayer.

Membrane protein structural biology – How far can the bugs take us? (Review)

Membrane proteins are core components of many essential cellular processes, and high-resolution structural data is therefore highly sought after. However, owing to the many bottlenecks associated with membrane protein crystallization, progress has been slow. One major problem is our inability to obtain sufficient quantities of membrane proteins for crystallization trials. Traditionally, membrane proteins have been isolated from natural sources, or for prokaryotic proteins, expressed by recombinant techniques. We are however a long way away from a streamlined overproduction of eukaryotic proteins. With this technical limitation in mind, we have probed the question as to how far prokaryotic homologues can take us towards a structural understanding of the eukaryotic/human membrane proteome(s).

Structural Biology of Membrane Proteins. Edited by Reinhard Grisshammer and Susan K. Buchanan.

Structural Biology of Membrane Proteins. Edited by Reinhard Grisshammer and Susan K. Buchanan.