Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.     

Extracellular Vesicles: A Platform for the Structure Determination of Membrane Proteins by Cryo-EM

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Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Protein Structure Prediction and Design in a Biologically Realistic Implicit Membrane

Protein design is a powerful tool for elucidating mechanisms of function and engineering new therapeutics and nanotechnologies. Although soluble protein design has advanced, membrane protein design remains challenging because of difficulties in modeling the lipid bilayer. In this work, we developed an implicit approach that captures the anisotropic structure, shape of water-filled pores, and nanoscale dimensions of membranes with different lipid compositions. The model improves performance in computational benchmarks against experimental targets, including prediction of protein orientations in the bilayer, ΔΔG calculations, native structure discrimination, and native sequence recovery. When applied to de novo protein design, this approach designs sequences with an amino acid distribution near the native amino acid distribution in membrane proteins, overcoming a critical flaw in previous membrane models that were prone to generating leucine-rich designs. Furthermore, the proteins designed in the new membrane model exhibit native-like features including interfacial aromatic side chains, hydrophobic lengths compatible with bilayer thickness, and polar pores. Our method advances high-resolution membrane protein structure prediction and design toward tackling key biological questions and engineering challenges.     

APBSmem: A Graphical Interface for Electrostatic Calculations at the Membrane

Electrostatic forces are one of the primary determinants of molecular interactions. They help guide the folding of proteins, increase the binding of one protein to another and facilitate protein-DNA and protein-ligand binding. A popular method for computing the electrostatic properties of biological systems is to numerically solve the Poisson-Boltzmann (PB) equation, and there are several easy-to-use software packages available that solve the PB equation for soluble proteins. Here we present a freely available program, called APBSmem, for carrying out these calculations in the presence of a membrane. The Adaptive Poisson-Boltzmann Solver (APBS) is used as a back-end for solving the PB equation, and a Java-based graphical user interface (GUI) coordinates a set of routines that introduce the influence of the membrane, determine its placement relative to the protein, and set the membrane potential. The software Jmol is embedded in the GUI to visualize the protein inserted in the membrane before the calculation and the electrostatic potential after completing the computation. We expect that the ease with which the GUI allows one to carry out these calculations will make this software a useful resource for experimenters and computational researchers alike. Three examples of membrane protein electrostatic calculations are carried out to illustrate how to use APBSmem and to highlight the different quantities of interest that can be calculated.     

A New Potential Model for Carbon Dioxide from AB Initio Calculations

Abstract

Ab initio quantum chemical calculations have been carried out for carbon dioxide dimer and the results have been used to establish potential functions usable in molecular simulations. Since the intermolecular interaction in carbon dioxide is fairly weak, careful treatment is required: this study uses 6–31G* basis set and takes electron correlations by the 2nd order Møller-Plesset theory into account. The potential energy surface is elucidated using the four representative relative configurations of the dimer. A new potential function model has been proposed on the basis of these ab initio data. In the super-critical region, this model is used to calculate the PVT relation of carbon dioxide fluid by the Monte Carlo simulations and confirmed to reproduce reasonably well the experimental isotherms.     

In Silico Analysis of Potential Outer Membrane Beta-Barrel Proteins in Aeromonas hydrophila Pangenome

International Journal of Peptide Research and Therapeutics - Outer membrane proteins (OMPs) of Aeromonas hydrophila have a variety of functional roles in virulence and pathogenesis and represent...     

The Advanced Properties of Circularized MSP Nanodiscs Facilitate High-resolution NMR Studies of Membrane Proteins

Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.     

The X-ray Structure of NccX from Cupriavidus metallidurans 31A Illustrates Potential Dangers of Detergent Solubilization When Generating and Interpreting Crystal Structures of Membrane Proteins

The x-ray structure of NccX, a type II transmembrane metal sensor, from Cupriavidus metallidurans 31A has been determined at a resolution of 3.12 Å. This was achieved after solubilization by dodecylphosphocholine and purification in the presence of the detergent. NccX crystal structure did not match the model based on the extensively characterized periplasmic domain of its closest homologue CnrX. Instead, the periplasmic domains of NccX appeared collapsed against the hydrophobic transmembrane segments, leading to an aberrant topology incompatible with membrane insertion. This was explained by a detergent-induced redistribution of the hydrophobic interactions among the transmembrane helices and a pair of hydrophobic patches keeping the periplasmic domains together in the native dimer. Molecular dynamics simulations performed with the full-length protein or with the transmembrane segments were used along with in vivo homodimerization assays (TOXCAT) to evaluate the determinants of the interactions between NccX protomers. Taken as a whole, computational and experimental results are in agreement with the structural model of CnrX where a cradle-shaped periplasmic metal sensor domain is anchored into the inner membrane by two N-terminal helices. In addition, they show that the main determinant of NccX dimerization is the periplasmic soluble domain and that the interaction between transmembrane segments is highly dynamic. The present work introduces a new crystal structure for a transmembrane protein and, in line with previous studies, substantiates the use of complementary theoretical and in vivo investigations to rationalize a three-dimensional structure obtained in non-native conditions.     

膜蛋白的拓扑学

膜蛋白的拓扑学是研究膜蛋白三维结构的出发点．利用融合蛋白和化学修饰等实验技术已确定了很多膜蛋白的拓扑学．对膜蛋白的转运与插膜的研究确定可能存在两类插膜元件．对已知拓扑学的膜蛋白的统计分析以及蛋白质工程的研究表明存在膜蛋白拓扑学的内正规则．目前已形成预测膜蛋白的拓扑学的比较可靠的策略,这在反向生物学上具有重要意义.但要进行三维结构的预测还有许多路要走．     

Basement Membrane Proteins: Structure,Assembly, and Cellular Interactions

Abstract

Basement membranes are thin layers of a specialized extracellular matrix that form the supporting structure on which epithelial and endothelial cells grow, and that surround muscle and fat cells and the Schwann cells of peripheral nerves. One common denominator is that they are always in close apposition to cells, and it has been well demonstrated that basement membranes do not only provide a mechanical support and divide tissues into compartments, but also influence cellular behavior. The major molecular constituents of basement membranes are collagen IV, laminin-entactin/nidogen complexes, and proteoglycans. Collagen IV provides a scaffold for the other structural macromolecules by forming a network via interactions between specialized N-and C-terminal domains. Laminin-entactin/nidogen complexes self-associate into less-ordered aggregates. These two molecular assemblies appear to be interconnected, presumably via binding sites on the entactin/nidogen molecule. In addition, proteoglycans are anchored into the membrane by an unknown mechanism, providing clusters of negatively charged groups. Specialization of different basement membranes is achieved through the presence of tissue-specific isoforms of laminin and collagen IV and of particular proteoglycan populations, by differences in assembly between different membranes, and by the presence of accessory proteins in some specialized basement membranes. Many cellular responses to basement membrane proteins are mediated by members of the integrin class of transmembrane receptors. On the intracellular side some of these signals are transmitted to the cytoskeleton, and result in an influence on cellular behavior with respect to adhesion, shape, migration, proliferation, and differentiation. Phosphorylation of integrins plays a role in modulating their activity, and they may therefore be a part of a more complex signaling system.