Structural Properties Of Membrane Proteins Research Articles

Challenges and Solutions in the Recombinant Expression of Membrane Proteins.

Membrane proteins are important components of the proteome and play key roles in many biological processes, such as signal transduction, material transport, cell recognition, etc. Membrane proteins are involved in several fields, and more and more researchers want to understand them. However, the structural properties of membrane proteins make their recombinant expression yield low. This adversely affects the study of the structure and function of membrane proteins. Therefore, it is crucial to have a comprehensive and up-to-date understanding of membrane protein recombinant expression. Based on the current stage of research on membrane proteins, the article describes the current challenges faced by membrane protein recombinant expression and the solutions that can be applied to lay the foundation for a better study of membrane proteins in the future.

Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membrane Proteins in Detergents.

There are very few generic methods to assess the stability and functional properties of membrane proteins solubilized in detergent. For this purpose, a thiol-reactive fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) can be used. An unfolding profile is obtained when the fluorochrome becomes fluorescent on reaction with cysteine residues that have been exposed during thermal denaturation of the protein population. The method was initially developed to optimize the stability of membrane proteins for crystallization studies, but in the course of our work we found many other applications. First, the assay can be used to study the binding of inhibitors, substrates, lipids, and other effectors to membrane proteins. Second, the assay can be used to understand the dynamics of proteins, allowing states to be defined by changes in accessibility of cysteine residues or by changes in specific amino acid interactions. Finally, the assay can be used to study state-dependent domain interactions, for example, as part of regulatory mechanisms. The CPM thermostability assay represents a broadly applicable and versatile tool for a wide range of applications in the functional and structural analysis of membrane proteins.

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes

Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na+/H+ antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na+/H+ antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na+/H+ antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.

Recent innovations in membrane-protein structural biology.

Innovations are expanding the capabilities of experimental investigations of the structural properties of membrane proteins. Traditionally, three-dimensional structures have been determined by measuring x-ray diffraction using protein crystals with a size of least 100 μm. For membrane proteins, achieving crystals suitable for these measurements has been a significant challenge. The availabilities of micro-focus x-ray beams and the new instrumentation of x-ray free-electron lasers have opened up the possibility of using submicrometer-sized crystals. In addition, advances in cryo-electron microscopy have expanded the use of this technique for studies of protein crystals as well as studies of individual proteins as single particles. Together, these approaches provide unprecedented opportunities for the exploration of structural properties of membrane proteins, including dynamical changes during protein function.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

The integration of membrane proteins into “lipid raft” membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Probing Structural Properties of KCNE1 Membrane Protein: A Site-Directed Spin Labeling EPR Study

KCNE1 is a single-transmembrane protein found in heart that modulates the activity of the KCNQ1 voltage-gated potassium channel. KCNE1 is very important for proper cardiac function. However, the structure of KCNE1 in a more native membrane environment is not completely understood. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy has emerged as a well-established method to study the structural properties of membrane proteins. In this study we have used continuous wave EPR and pulsed EPR techniques in combination with the SMA-Lipodisq nanoparticles based sample preparation to obtain more accurate and precise EPR data to answer pertinent structural questions on KCNE1. CW-EPR and DEER data were collected on several single and double spin labeling sites on KCNE1 in micelles, proteoliposomes and lipodisqs. We obtained a significant improvement in the quality of EPR measurements in lipodisq samples compared to proteoliposomes. The structural and dynamic properties of KCNE1 will be discussed.

Utilizing Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy to Probe the Structure of Membrane Proteins

New approaches are needed to more efficiently probe the structural properties of membrane proteins. A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed Electron Paramagnetic Resonance (EPR) technique of Electron Spin Echo Envelope Modulation (ESEEM). This technique can measure short-range distances between a nitroxide spin label and a 2H nucleus out to approximately 8Å. For this study a model membrane peptide M2δ, was constructed by solid phase peptide synthesis and inserted into a DMPC/DHPC bicelle membrane. We report for the first time, the direct detection of 2H modulation between a 2H-labeled d8 Val residue and a nitroxide spin label three and four residues away that is characteristic of an alpha-helical secondary structure. Simulations of the ESEEM data reveal a distance of approximately 6.4 +/- 0.5Å that agrees well with molecular modeling studies. ESEEM spectra in this work yielded high-quality data in less than an hour with as little as 35μg of protein sample.

Site-Directed Mutagenesis Combined with Oxidative Methionine Labeling for Probing Structural Transitions of a Membrane Protein by Mass Spectrometry

Exposure of the membrane protein bacteriorhodopsin (BR) to SDS induces partial breakdown of the native conformation. The exact structural properties of this SDS state remain a matter of debate, despite its widespread use in BR folding experiments. The current work employs hydroxyl radical (·OH) labeling in conjunction with mass spectrometry (MS)-based peptide mapping for probing the solvent accessibility of individual BR segments in the presence of SDS. Previous work revealed methionine sulfoxide formation to be the dominant oxidative pathway. Those data suggested extensive unfolding of helices A and D in SDS. Unfortunately, the lack of Met residues in helices C and F implies that no direct information on the behavior of the latter two elements could be obtained. Here, we address this problem by employing two variants with additional Met residues, L93M (helix C) and V179M (helix F). The oxidation behavior of the resulting 11 methionines can be grouped into three categories: (1) extensively labeled both in native BR and in SDS (loop residues M32, M68, and M163); (2) protected in the native state but not in SDS (M20, M118); (3) always protected (M56, M60, M93, M145, M179, M209). These data show that a solvent-inaccessible core is retained in SDS. This core consists of partially intact helices B, C, E, F, and G. The termini of these helices are highly dynamic and/or unraveled, particularly on the cytoplasmic side. Overall, this work demonstrates how the use of engineered ·OH labeling sites can provide insights into structural properties of membrane proteins.

EPR Spectroscopic Studies on the Structural and Dynamic Properties of Human KCNE1 Membrane Protein in Lipid Bilayers

The KCNE1 membrane protein regulates KCNQ1, which forms the voltage-gated potassium channel in the human heart. Mutations in these genes are responsible for the human genetic disease, long QT syndrome. However, the structure of KCNE1 in a lipid bilayer and biophysical basis for KCNE1's modulation of KCNQ1 are not completely understood. Recent research from Sanders group using solution NMR indicates the transmembrane domain of KCNE1 is a curved alpha-helix and is flanked by intra- and extracellular domains comprised of alpha-helices joined by flexible linkers (Kang et al., Biochemistry 2008 47:7999-8006). Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy has emerged as a well-established method to study the structural properties of membrane proteins. Our objective is to use advanced EPR spectroscopic techniques including CW and pulsed EPR spectroscopy to obtain structural parameters of the KCNE1 protein in a bilayer. To date, we have successfully over-expressed KCNE1 in E. coli and reconstituted the protein into POPG/POPC lipid bilayers. Electrophysiological experiments confirm KCNE1 can co-assemble with the channel protein, KCNQ1 and are fully functional. Electrophysiological experiments further confirm that KCNE1 is inserted correctly into the lipid-bilayers. Additionally we have also demonstrated potential use of DEER (Double Electron-Electron Resonance) and CW-EPR power saturation experiments for distance and depth measurements of KCNE1 in lipid bilayers, respectively.

Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation.

The dynamic and structural properties of membrane proteins are intimately affected by the lipid bilayer. One property of membrane proteins is uniaxial rotational diffusion, which depends on the membrane viscosity and thickness. This rotational diffusion is readily manifested in solid-state NMR spectra as characteristic line shapes and temperature-dependent line narrowing or broadening. We show here that this whole-body uniaxial diffusion is suppressed in lipid bilayers mimicking the composition of eukaryotic cell membranes, which are rich in cholesterol and sphingomyelin. We demonstrate this membrane-induced immobilization on the transmembrane peptide of the influenza A M2 (AM2-TM) proton channel protein. At physiological temperature, AM2-TM undergoes uniaxial diffusion faster than approximately 10(5) s(-1) in DLPC, DMPC, and POPC bilayers, but the motion is slowed by 2 orders of magnitude, to <10(3) s(-1), in a cholesterol-rich virus envelope-mimetic membrane ("viral membrane"). The immobilization is manifested as near rigid-limit (2)H quadrupolar couplings and (13)C-(1)H, (15)N-(1)H, and (13)C-(15)N dipolar couplings for all labeled residues. The immobilization suppresses intermediate time scale broadening of the NMR spectra, thus allowing high-sensitivity and high-resolution spectra to be measured at physiological temperature. The conformation of the protein in the viral membrane is more homogeneous than in model PC membranes, as evidenced by the narrow (15)N lines. The immobilization of the M2 helical bundle by the membrane composition change indicates the importance of studying membrane proteins in environments as native as possible. It also suggests that eukaryote-mimetic lipid membranes may greatly facilitate structure determination of membrane proteins by solid-state NMR.

2P-078 Prediction of equilibrium and dynamic structural properties of membrane proteins(Membrane proteins,The 47th Annual Meeting of the Biophysical Society of Japan)

2P-078 Prediction of equilibrium and dynamic structural properties of membrane proteins(Membrane proteins,The 47th Annual Meeting of the Biophysical Society of Japan)

3P-099 膜蛋白質の1次元構造属性の予測(膜蛋白質,第46回日本生物物理学会年会)

3P-099 膜蛋白質の1次元構造属性の予測(膜蛋白質,第46回日本生物物理学会年会)

Interactions of β-Lactoglobulin and α-Lactalbumin with Lipids: A Review

Although of excellent nutritional quality, whey proteins are underutilized. To provide new insights for their potential in food products, interactions of α-lactalbumin and β-lactoglobulin with lipids are reviewed. Structural properties of membrane proteins and methods for predicting interactions of proteins with lipids are discussed briefly with regard to lipid-protein complexes in foods. Effects of surfactants on conformations of α-lactalbumin and β-lactoglobulin also are presented. Studies of the interactions of these proteins with synthetic lipids in monolayers and single bilayer vesicles are described. Implications of these studies on the use of whey protein in food emulsions are considered.