Determining membrane protein structures: still a challenge! (original) (raw)
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A Method to Assess Correct/Misfolded Structures of Transmembrane Domains of Membrane Proteins
Motivation: Integral membrane proteins (MP) are pharmaceutical targets of exceptional importance since more than 50 % of currently marketed drugs target these objects. Due to technical difficulties, modern experimental methods often fail to determine 3D structure of MPs. Computational methods for modeling MPs structure and assessment of these models' quality may be very helpful in this case. Results: We propose a novel method for quantitative estimation of the transmembrane (TM) domains models' quality. The approach is based on the concept of environmental profile. A non-redundant set of 26 high-resolution X-ray structures of α-helical TM domains is used to define five classes of residues' environment, considering polarity of nearest protein surrounding and accessibility for a given residue. Residues' preferences for each environment class are calculated. The main results are: (1) The proteins length correlates with the proposed scoring function values, defining a way to differentiate "well-folded" structures from misfolded ones; (2) The method efficiently delineates crystallographic structure of visual rhodopsin both in a set of twelve its computer models, containing certain errors and ensemble of artificially generated misfolded structures of rhodopsin; (3) Photosynthetic MPs demonstrate different score-length dependency, suggesting distinct packing characteristics for these proteins.
Bioinformatics (Oxford, England), 2016
The experimental determination of membrane protein orientation within the lipid bilayer is extremely challenging, such that computational methods are most often the only solution. Moreover, obtaining all-atom 3D structures of membrane proteins is also technically difficult, and many of the available data are either experimental low-resolution structures or theoretical models, whose structural quality needs to be evaluated. Here, to address these two crucial problems, we propose OREMPRO, a web server capable of both (i) positioning a-helical and b-sheet transmembrane domains in the lipid bilayer and (ii) assessing their structural quality. Most importantly, OREMPRO uses the sole alpha carbon coordinates, which makes it the only web server compatible with both high and low structural resolutions. Finally, OREMPRO is also interesting in its ability to process coarse-grained protein models, by using coordinates of backbone beads in place of alpha carbons.
Protein Science, 2007
Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins.
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.
Molecules
In eukaryotic cells, membrane proteins play a crucial role. They fall into three categories: intrinsic proteins, extrinsic proteins, and proteins that are essential to the human genome (30% of which is devoted to encoding them). Hydrophobic interactions inside the membrane serve to stabilize integral proteins, which span the lipid bilayer. This review investigates a number of computational and experimental methods used to study membrane proteins. It encompasses a variety of technologies, including electrophoresis, X-ray crystallography, cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance spectroscopy (NMR), biophysical methods, computational methods, and artificial intelligence. The link between structure and function of membrane proteins has been better understood thanks to these approaches, which also hold great promise for future study in the field. The significance of fusing artificial intelligence with experimental data to improve our comprehension of membrane p...
Probing the Transmembrane Structure and Topology of
2013
Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.
Transmembrane domains interactions within the membrane milieu: Principles, advances and challenges
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2012
Protein-protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane-embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD-TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD-TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD-TMD interactions allows for the design of novel short D-and L-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Where do we go from here? Membrane protein research beyond the structure-function horizon
Biochimica Et Biophysica Acta - Biomembranes, 2018
Where do we go from here? Membrane protein research beyond the structure-function horizon Biology has evolved and thrives in an aqueous environment. The water-insolubility of membrane proteins is one of the biggest reasons why membrane protein research has been among the most challenging aspects of protein biology, with every study being plagued by uncertainties regarding the physiological relevance of its findings. But thanks to savvy technical innovations (see below), researchers are closer than ever to getting a firm handle on the biology of several membrane proteins, even to the point where bench-to-bedside research is becoming a consideration. This Special Issue is dedicated to such advances and is our humble attempt at highlighting some key areas. For a while now, the workflow of membrane protein research has proceeded as follows: (i) A select protein of interest is cloned out from its native genome, and tested in a battery of functional biochemical assays, either in a convenient heterologous host or in purified form. (ii) Mutations are made across the protein to assess their various effects, and predictions are made about its structure and mechanism(s). In the past few decades, this cycle was iterated several times over with many discoveries of novel classes of membrane proteins being made, based on topology mappings and functional nuances. The advances in biophysical tools in the form of high-resolution X-ray and cryo-electron microscopy (cryoEM) structures have allowed the addition of architecture to be ascribed to protein function. However, for the longest time, obtaining the structure of a researcher's favorite protein (especially if it happened to be a membrane protein) was the culmination of a lifetime's worth of workif it was ever attained. Membrane protein research certainly has changed, and although the first high-resolution structure of a membrane protein by cryoEM was only published four years ago [1], the resulting avalanche of high-resolution cryoEM membrane protein structures certainly profits from the fact that their structure-function relationships had been heavily investigated for decades. It is not a stretch, then, to expect to see protein structures for all major membrane protein families, and potentially all medically relevant targets in the next couple of years. But where do we go from there? What is left to do and what can structures fall short of revealing? In the first paper in this Special Issue, Seeger muses on how it is time for us to reflect on what we, as a field, can do in "times of countless structures" [2]. And encouragingly, he reminds us that structural biology will not become obsolete any time soon. Instead, a new species of structural biologist is emerging-that of an integrated scientist, who works with experts on the functional, computational, and biophysical facets of membrane proteins to arrive at a holistic understanding for each protein of interest. Advances in biophysical tools have, in close collaboration with high resolution structures and functional data, provided means to detect interatomic distance changes in membrane-embedded proteins, to assess their dynamics and now even allow to predict their behavior to a certain degree through molecular dynamic simulations of proteins in modelled native-like environments. But no matter how advanced our techniques become, ultimately, they still rely on a careful sample preparation. And while it may by now seem reasonably straight forward to pull a membrane protein out of its lipid environment to then reconstitute it back into a lipid bilayer, there has also been a huge demand for optimizing these approaches and obtain more native-like reconstitution conditions. The introduction of nanodiscs clearly revolutionized the field (e.g. [3-5]) as they allow to study membrane proteins in near-native conditions. Another novel technology for the reconstitution of membrane proteins for functional studies using styrene-maleic acid copolymer discs is discussed in Pollock et al. [6]. Here, a major advantage is that the protein can be purified in its native membrane, thus maintaining lipid composition and presumably, at least for a short amount of time, asymmetry. Together, combining advances in protein preparation with biophysical approaches do a fantastic job validating and accelerating functional experimentation much further than would otherwise have been possible with the individual techniques alone. As spectroscopic techniques become more advanced, they begin to shed light on intricate details of protein dynamics as described by Spadaccini et al., using solid-state NMR (nuclear magnetic resonance) in combination with DNP (dynamic nuclear polarization) [7], Zhogbi et al., using LRET (luminescence resonance electron transfer) [8] and Bordignon and Bleicken with EPR (electron paramagnetic resonance) [9]. EPR spectroscopy has long been recognized as a formidable tool to study membrane protein dynamics, and in the present review, the authors also give an overview over recent hardware, pulse sequence and spin label improvements. EPR studies are often combined with NMR studies [10,11], as global and local conformational changes can be studied in a complementary fashion. DNP-enhanced NMR additionally allows spectra to be recorded in a fraction of the previously required time, thus making lowly populated conformations or difficult to obtain samples accessible to inherently insensitive NMR studies. Spadaccini et al. show how substrate interactions with MsbA lead to specific changes in helix 4 and 6 of this ABC transporter [7]. Likewise, Zhogbi et al. use this ABC transporter to demonstrate the advantages of LRET in the study of membrane proteins, such as the long lifetime of excited states for the lanthanide donor and the line spectra emissions [8].
Database : the journal of biological databases and curation, 2017
Knowing the position of protein structures within the membrane is crucial for fundamental and applied research in the field of molecular biology. Only few web resources propose coordinate files of oriented transmembrane proteins, and these exclude predicted structures, although they represent the largest part of the available models. In this article, we present TMPL (http://www.dsimb.inserm.fr/TMPL/), a database of transmembrane protein structures (α-helical and β-sheet) positioned in the lipid bilayer. It is the first database to include theoretical models of transmembrane protein structures, making it a large repository with more than 11 000 entries. The TMPL database also contains experimentally solved protein structures, which are available as either atomistic or coarse-grained models. A unique feature of TMPL is the possibility for users to update the database by uploading, through an intuitive web interface, the membrane assignments they can obtain with our recent OREMPRO web ...
Biophysical Journal, 2014
The membrane environment, its composition, dynamics, and remodeling, have been shown to participate in the function and organization of a wide variety of transmembrane (TM) proteins, making it necessary to study the molecular mechanisms of such proteins in the context of their membrane settings. We review some recent conceptual advances enabling such studies, and corresponding computational models and tools designed to facilitate the concerted experimental and computational investigation of protein-membrane interactions. To connect productively with the high resolution achieved by cognate experimental approaches, the computational methods must offer quantitative data at an atomistically detailed level. We show how such a quantitative method illuminated the mechanistic importance of a structural characteristic of multihelical TM proteins, that is, the likely presence of adjacent polar and hydrophobic residues at the protein-membrane interface. Such adjacency can preclude the complete alleviation of the well-known hydrophobic mismatch between TM proteins and the surrounding membrane, giving rise to an energy cost of residual hydrophobic mismatch. The energy cost and biophysical formulation of hydrophobic mismatch and residual hydrophobic mismatch are reviewed in the context of their mechanistic role in the function of prototypical members of multihelical TM protein families: 1), LeuT, a bacterial homolog of mammalian neurotransmitter sodium symporters; and 2), rhodopsin and the b1and b2-adrenergic receptors from the G-protein coupled receptor family. The type of computational analysis provided by these examples is poised to translate the rapidly growing structural data for the many TM protein families that are of great importance to cell function into ever more incisive insights into mechanisms driven by protein-ligand and protein-protein interactions in the membrane environment.