Positioning of proteins in membranes: a computational approach

A new computational approach has been developed to determine the spatial arrangement of proteins in membranes by minimizing their transfer energies from water to the lipid bilayer. The membrane hydrocarbon core was approximated as a planar slab of adjustable thickness with decadiene-like interior and interfacial polarity profiles derived from published EPR studies. Applicability and accuracy of the method was verified for a set of 24 transmembrane proteins whose orientations in membranes have been studied by spin-labeling, chemical modification, fluorescence, ATR FTIR, NMR, cryo-microscopy, and neutron diffraction. Subsequently, the optimal rotational and translational positions were calculated for 109 transmembrane, five integral monotopic and 27 peripheral protein complexes with known 3D structures. This method can reliably distinguish transmembrane and integral monotopic proteins from water-soluble proteins based on their transfer energies and membrane penetration depths. The accuracies of calculated hydrophobic thicknesses and tilt angles were approximately 1 A and 2 degrees, respectively, judging from their deviations in different crystal forms of the same proteins. The hydrophobic thicknesses of transmembrane proteins ranged from 21.1 to 43.8 A depending on the type of biological membrane, while their tilt angles with respect to the bilayer normal varied from zero in symmetric complexes to 26 degrees in asymmetric structures. Calculated hydrophobic boundaries of proteins are located approximately 5 A lower than lipid phosphates and correspond to the zero membrane depth parameter of spin-labeled residues. Coordinates of all studied proteins with their membrane boundaries can be found in the Orientations of Proteins in Membranes (OPM) database:http://opm.phar.umich.edu/.     

Transmembrane proteins in the Protein Data Bank: identification and classification

MOTIVATION: Integral membrane proteins play important roles in living cells. Although these proteins are estimated to constitute 25% of proteins at a genomic scale, the Protein Data Bank (PDB) contains only a few hundred membrane proteins due to the difficulties with experimental techniques. The presence of transmembrane proteins in the structure data bank, however, is quite invisible, as the annotation of these entries is rather poor. Even if a protein is identified as a transmembrane one, the possible location of the lipid bilayer is not indicated in the PDB because these proteins are crystallized without their natural lipid bilayer, and currently no method is publicly available to detect the possible membrane plane using the atomic coordinates of membrane proteins. RESULTS: Here, we present a new geometrical approach to distinguish between transmembrane and globular proteins using structural information only and to locate the most likely position of the lipid bilayer. An automated algorithm (TMDET) is given to determine the membrane planes relative to the position of atomic coordinates, together with a discrimination function which is able to separate transmembrane and globular proteins even in cases of low resolution or incomplete structures such as fragments or parts of large multi chain complexes. This method can be used for the proper annotation of protein structures containing transmembrane segments and paves the way to an up-to-date database containing the structure of all known transmembrane proteins and fragments (PDB_TM) which can be automatically updated. The algorithm is equally important for the purpose of constructing databases purely of globular proteins.     

Simulation of NMR data from oriented membrane proteins: practical information for experimental design.

Several hundred solid state NMR dipolar couplings and chemical shift anisotropies were simulated for the polytopic membrane protein, bacteriorhodopsin, and for an idealized transmembrane peptide conforming to several different secondary structures (alpha- and 3(10)-helices and parallel and antiparallel beta-sheets), each at several tilt angles with respect to the bilayer normal. The use of macroscopically oriented samples was assumed. The results of these simulations suggest: (i) Because of the r-3 dependence of dipolar coupling, it is likely to prove difficult to successfully execute uniform isotopic enrichment strategies to generate large numbers of quantitatively interpretable structural measurements in oriented sample NMR studies of membrane proteins. (ii) There are a number of readily implementable specific isotopic labeling schemes which can yield data patterns sufficient to identify local secondary structure for transmembrane segments of idealized proteins which are tilted by < 10 degrees with respect to the bilayer normal. (iii) The measurement of dipolar coupling constants between 13C-, 19F-, and/or 3H-labeled side chains of proximal residues may prove effective as routes to long range tertiary structural data constraints.     

How protein transmembrane segments sense the lipid environment

Integral membrane proteins have central roles in a vast number of vital cellular processes. A structural feature that most membrane proteins have in common is the presence of one or more alpha-helices with which they traverse the lipid bilayer. Because of the interaction with the surrounding lipids, the organization of these transmembrane helices will be sensitive to lipid properties like lateral packing, hydrophobic thickness, and headgroup charge. The helices may adapt to the lipids in different ways, which in turn can influence the structure and function of the intact membrane protein. In this review, we will focus on how the lipid environment influences two specific properties of transmembrane segments: their lateral association and their tilt with respect to the bilayer normal.     

Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Biophysical properties of lipids and dynamic membranes

The lipid bilayer is a 3D assembly with a rich variety of physical features that modulate cell signaling and protein function. Lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched and as new constituents are added, removed or chemically modified. Recent studies have revealed how differences in structure between the two leaflets of the bilayer and between different areas of the bilayer can interact together with membrane deformation to alter the activities of transmembrane channels and peripheral membrane binding proteins. Here, we highlight some recent reports that the physical properties of the membrane can help control the function of transmembrane proteins and the motor-dependent elongation of internal organelles, such as the endoplasmic reticulum.     

TMDET: web server for detecting transmembrane regions of proteins by using their 3D coordinates

The structure of integral membrane proteins is determined in the absence of the lipid bilayer; consequently the membrane localization of the protein is usually not specified in the corresponding PDB file. Recently, we have developed a new method called TMDET which determines the most possible localization of the membrane relative to the protein structure, and gives the annotation of the membrane embedded parts of the sequence. The entire Protein Data Bank has been scanned by the new TMDET algorithm resulting in the database of structurally determined transmembrane proteins (PDB_TM). Here we present the web interface of the TMDET algorithm to allow scientists to determine the membrane localization of structural data prior to deposition or to analyze model structures.     

Membrane protein insertase YidC in bacteria and archaea

The insertion of proteins into the prokaryotic plasma membrane is catalyzed by translocases and insertases. On one hand, the Sec translocase operates as a transmembrane channel that can open laterally to first bind and then release the hydrophobic segments of a substrate protein into the lipid bilayer. On the other hand, YidC insertases interact with their substrates in a groove‐like structure at an amphiphilic protein–lipid interface thus allowing the transmembrane segments of the substrate to slide into the lipid bilayer. The recently published high‐resolution structures of YidC provide new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane.     

Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix

The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

E(z), a depth-dependent potential for assessing the energies of insertion of amino acid side-chains into membranes: derivation and applications to determining the orientation of transmembrane and interfacial helices

We have developed an empirical residue-based potential (E(z) potential) for protein insertion in lipid membranes. Propensities for occurrence as a function of depth in the bilayer were calculated for the individual amino acid types from their distribution in known structures of helical membrane proteins. The propensities were then fit to continuous curves and converted to a potential using a reverse-Boltzman relationship. The E(z) potential demonstrated a good correlation with experimental data such as amino acid transfer free energy scales (water to membrane center and water to interface), and it incorporates transmembrane helices of varying composition in the membrane with trends similar to those obtained with translocon-mediated insertion experiments. The potential has a variety of applications in the analysis of natural membrane proteins as well as in the design of new ones. It can help in calculating the propensity of single helices to insert in the bilayer and estimate their tilt angle with respect to the bilayer normal. It can be utilized to discriminate amphiphilic helices that assume a parallel orientation at the membrane interface, such as those of membrane-active peptides. In membrane protein design applications, the potential allows an environment-dependent selection of amino acid identities.     

Membrane-protein integration and the role of the translocation channel

Most eukaryotic membrane proteins are integrated into the lipid bilayer during their synthesis at the endoplasmic reticulum (ER). Their integration occurs with the help of a protein-conducting channel formed by the heterotrimeric Sec61 membrane-protein complex. The crystal structure of an archaeal homolog of the complex suggests mechanisms that enable the channel to open across the membrane and to release laterally hydrophobic transmembrane segments of nascent membrane proteins into lipid. Many aspects of membrane-protein integration remain controversial and poorly understood, but new structural data provide testable hypotheses. We propose a model of how the channel recognizes transmembrane segments, orients them properly with respect to the plane of the membrane and releases them into lipid. We also discuss how the channel would prevent small molecules from crossing the lipid bilayer while it is integrating proteins.     

High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-beta-maltoside or dodecyl-beta-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20 degrees C and at 4 degrees C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.     

Lipid-protein interactions

Intrinsic membrane proteins are solvated by a shell of lipid molecules interacting with the membrane-penetrating surface of the protein; these lipid molecules are referred to as annular lipids. Lipid molecules are also found bound between transmembrane α-helices; these are referred to as non-annular lipids. Annular lipid binding constants depend on fatty acyl chain length, but the dependence is less than expected from models based on distortion of the lipid bilayer alone. This suggests that hydrophobic matching between a membrane protein and the surrounding lipid bilayer involves some distortion of the transmembrane α-helical bundle found in most membrane proteins, explaining the importance of bilayer thickness for membrane protein function. Annular lipid binding constants also depend on the structure of the polar headgroup region of the lipid, and hotspots for binding anionic lipids have been detected on some membrane proteins; binding of anionic lipid molecules to these hotspots can be functionally important. Binding of anionic lipids to non-annular sites on membrane proteins such as the potassium channel KcsA can also be important for function. It is argued that the packing preferences of the membrane-spanning α-helices in a membrane protein result in a structure that matches nicely with that of the surrounding lipid bilayer, so that lipid and protein can meet without either having to change very much.     

Jumping to rafts: gatekeeper role of bilayer elasticity

Two of the physiologically important processes that take place in biological membranes are the partitioning of water-soluble proteins into the membrane and the sequestering of specific transmembrane proteins into membrane microdomains or 'rafts'. Although these two processes often involve different classes of protein, recent biophysical studies indicate that they both strongly depend on the structural and elastic properties of the membrane bilayer. That is, both the partitioning of peptides into membranes and the distribution of transmembrane peptides in the plane of the membrane are modulated by physical properties of the lipid bilayer that are controlled by cholesterol content and the composition of the phospholipid hydrocarbon chain.     

TOPDOM: database of domains and motifs with conservative location in transmembrane proteins

The TOPDOM database is a collection of domains and sequence motifs located consistently on the same side of the membrane in alpha-helical transmembrane proteins. The database was created by scanning well-annotated transmembrane protein sequences in the UniProt database by specific domain or motif detecting algorithms. The identified domains or motifs were added to the database if they were uniformly annotated on the same side of the membrane of the various proteins in the UniProt database. The information about the location of the collected domains and motifs can be incorporated into constrained topology prediction algorithms, like HMMTOP, increasing the prediction accuracy. AVAILABILITY: The TOPDOM database and the constrained HMMTOP prediction server are available on the page http://topdom.enzim.hu CONTACT: tusi@enzim.hu; lkalmar@enzim.hu.     

Membrane lipids and transporter function

Transport proteins are essential for cells in allowing the exchange of substances between cells and their environment across the lipid bilayer forming a tight barrier. Membrane lipids modulate the function of transmembrane proteins such as transporters in two ways: Lipids are tightly and specifically bound to transport proteins and in addition they modulate from the bulk of the lipid bilayer the function of transport proteins. This overview summarizes currently available information at the ultrastructural level on lipids tightly bound to transport proteins and the impact of altered bulk membrane lipid composition. Human diseases leading to altered lipid homeostasis will lead to altered membrane lipid composition, which in turn affect the function of transporter proteins.     

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.     

Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding. A site-directed spin labeling study

The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.     

Robust Driving Forces for Transmembrane Helix Packing

The packing structures of transmembrane helices are traditionally attributed to patterns in residues along the contact surface. In this view, besides keeping the helices confined in the membrane, the bilayer has only a minor effect on the helices structure. Here, we use two different approaches to show that the lipid environment has a crucial effect in determining the cross-angle distribution of packed helices. We analyzed structural data of a membrane proteins database. We show that the distribution of cross angles of helix pairs in this database is statistically indistinguishable from the cross-angle distribution of two noninteracting helices imbedded in the membrane. These results suggest that the cross angle is, to a large extent, determined by the tilt angle of the individual helices. We test this hypothesis using molecular simulations of a coarse-grained model that contains no specific residue interactions. These simulations reproduce the same cross-angle distribution as found in the database. As the tilt angle of a helix is dominated by hydrophobic mismatch between the protein and surrounding lipids, our results indicate that hydrophobic mismatch is the dominant factor guiding the transmembrane helix packing. Other short-range forces might then fine-tune the structure to its final configuration.