Crystal Structures Of Membrane Proteins Research Articles

Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g- conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Ogamma atom of Ser and Thr and the backbone carbonyl oxygen at position i-3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the beta(2)-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.

“Torsional tapping” atomic force microscopy using T-shaped cantilevers

Torsional oscillation of atomic force microscope cantilevers has been shown to offer increased optical lever sensitivity, quality factor, resonant frequency, and stiffness as compared to flexural oscillation. In this letter, T-shaped cantilevers are oscillated torsionally to give a tapping motion at the tip. This gives many of the advantages of small cantilevers, without the requirement for specialized detection optics. In order to demonstrate the capability of this technique, high resolution images of LH2 membrane protein crystal structures are presented. Reduced settle time and tip-sample force under error signal are also demonstrated.

Filling the Gap: A streamlined approach for monitoring expression and purification of membrane proteins with a periplasmic C‐terminus via GFP fluorescence

Crystal structures of membrane proteins are notoriously difficult to obtain. These proteins reside in phospholipid bilayers making them difficult to express, purify, stabilize and structurally resolve. Previously, inner membrane proteins (IMP) have been recombinantly expressed and monitored using a C‐terminal green fluorescent protein (GFP) fusion in Escherichia coli. This method proved extremely useful, but is only effective for IMP with C‐termini localizing to the cytoplasm (CI), where the GFP folds correctly. To fill the gap for IMP with C‐termini localizing to the periplasm (CO), we developed the expression vectors: pWarf(‐) and pWarf(+). While pWarf(‐) fuses GFP to the C‐terminus of the IMP, pWarf(+) fuses the single transmembrane helix protein Glycophorin A and subsequent GFP to the IMP. The pWarf(+) vector effectively converts CO IMP to CI IMP. Eleven E. coli IMP from three families were selected for their predicted CO topology. High level whole cell fluorescence was observed only in the pWarf(+) system. Using the pWarf(+) vector, overexpression was rapidly screened, detergents were selected by fluorescent size exclusion chromatography and stability was monitored. Large scale purification is in progress for crystallization trials. By developing the pWarf vector system, a comprehensive, streamlined approach to monitoring overexpression, purification and stabilization has been achieved for IMP. Funding from AHA.

Position of helical kinks in membrane protein crystal structures and the accuracy of computational prediction

The structural features of helical transmembrane (TM) proteins, such as helical kinks, tilts, and rotational orientations are important in modulation of their function and these structural features give rise to functional diversity in membrane proteins with similar topology. In particular, the helical kinks caused by breaking of the backbone hydrogen bonds lead to hinge bending flexibility in these helices. Therefore it is important to understand the nature of the helical kinks and to be able to reproduce these kinks in structural models of membrane proteins. We have analyzed the position and extent of helical kinks in the transmembrane helices of all the crystal structures of membrane proteins taken from the MPtopo database, which are about 405 individual helices of length between 19 and 35 residues. 44% of the crystal structures of TM helices showed a significant helical kink, and 35% of these kinks are caused by prolines. Many of the non-proline helical kinks are caused by other residues like Ser and Gly that are located at the center of helical kinks. The side chain of Ser makes a hydrogen bond with the main chain carbonyl of the i − 4th or i + 4th residue thus making a kink. We have also studied how well molecular dynamics (MD) simulations on isolated helices can reproduce the position of the helical kinks in TM helices. Such a method is useful for structure prediction of membrane proteins. We performed MD simulations, starting from a canonical helix for the 405 TM helices. 1 ns of MD simulation results show that we can reproduce about 79% of the proline kinks, only 59% of the vestigial proline kinks and 18% of the non-proline helical kinks. We found that similar results can be obtained from choosing the lowest potential energy structure from the MD simulation. 4–14% more of the vestigial prolines were reproduced by replacing them with prolines before performing MD simulations, and changing the amino acid back to proline after the MD simulations. From these results we conclude that the position of the helical kinks is dependent on the TM sequence. However the extent of helical kinking may depend on the packing of the rest of the protein and the lipid bilayer.

SIBAR Descriptors and Support Vector Machine for ABCB1 Substrate Prediction

As part of the ATP-binding cassette transporter superfamily ABCB1 (P-gp) exports a multitude of xenobiotics and is strongly connected to multi-drug resistance (MDR). With the decreasing number of new drugs entering the market prediction of possible side effects in new lead structures gains increasing importance. In silico methods hereby have become highly significant. As crystal structures of membrane proteins are difficult to produce and a structure of ABCB1 has yet to be published, ligand based approaches are still the method of choice. The aim of this study is to compare two approaches widely used for substrate prediction.

Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease.

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function.

Structure and mechanism of the lactose permease

More than 20% of the genes sequenced thus far appear to encode polytopic transmembrane proteins involved in a multitude of critical functions, particularly energy and signal transduction. Many are important with regard to human disease (e.g., depression, diabetes, drug resistance), and many drugs are targeted to membrane transport proteins (e.g., fluoxetine and omeprazole). However, the number of crystal structures of membrane proteins, especially ion-coupled transporters, is very limited. Recently, an inward-facing conformer of the Escherichia coli lactose permease (LacY), a paradigm for the Major Facilitator Superfamily, which contains almost 4000 members, was solved at about 3.5 Å in collaboration with Jeff Abramson and So Iwata at Imperial College London. This intensively studied membrane transport protein is composed of two pseudo-symmetrical 6-helix bundles with a large internal cavity containing bound sugar and open to the cytoplasm only. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism is proposed in which the binding site is alternatively accessible to either side of the membrane. To cite this article: H.R. Kaback, C. R. Biologies 328 (2005).

How to read clinical journals: XII. How you too can profit from pharmaceutical advertisements

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (∼0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Position-dependence of stabilizing polar interactions of asparagine in transmembrane helical bundles.

Recent studies with model peptides and statistical analyses of the crystal structures of membrane proteins have shown that buried polar interactions contribute significantly to the stabilization of the three-dimensional structures of membrane proteins. Here, we probe how the location of these polar groups along the transmembrane helices affect their free energies of interaction. Asn residues were placed singly and in pairs at three positions within a model transmembrane helix, which had previously been shown to support the formation of trimers in micelles. The model helix was designed to form a transmembrane coiled coil, with Val side chains at the "a" positions of the heptad repeat. Variants of this peptide were prepared in which an Asn residue was introduced at one or more of the "a" positions, and their free energies of association were determined by analytical ultracentrifugation. When placed near the middle of the transmembrane helix, the formation of trimers was stabilized by at least -2.0 kcal/mol per Asn side chain. When the Asn was placed at the interface between the hydrophobic and polar regions of the peptide, the substitution was neither stabilizing nor destabilizing (0.0 +/- 0.5 kcal/mol of monomer). Finally, it has previously been shown that a Val-for-Asn mutation in a water-soluble coiled coil destabilizes the structure by approximately 1.5 kcal/mol of monomer [Acharya, A., et al. (2002) Biochemistry 41, 14122-14131]. Thus, the headgroup region of a micelle appears to have a conformational impact intermediate between that of bulk water and the apolar region of micelle. A similarly large dependence on the location of the polar residues was found in a statistical survey of helical transmembrane proteins. The tendency of different types of residues to be buried in the interiors versus being exposed to lipids was analyzed. Asn and Gln show a very strong tendency to be buried when they are located near the middle of a transmembrane helix. However, when placed near the ends of transmembrane helices, they show little preference for the surface versus the interior of the protein. These data show that Asn side chains within the apolar region of the transmembrane helix provide a significantly larger driving force for association than Asn residues near the apolar/polar interface. Thus, although polar interactions are able to strongly stabilize the folding of membrane proteins, the energetics of association depend on their location within the hydrophobic region of a transmembrane helix.

Channel Gating Theory Revised

Voltage-gated ion channels, which are opened (or gated) in response to changes in the voltage across the cell membrane, control fundamental signaling properties in cells, particularly in neurons and muscle cells. A great deal of effort has been expended in trying to understand the molecular basis for this behavior. A sensor region of voltage-gated K + channels has been defined and is known to move across the membrane to promote channel gating. Two papers from Jiang and colleagues in the MacKinnon laboratory provide new clues by revealing the structure of the voltage sensor and characterizing its movement in the membrane. X-ray crystal structures of membrane proteins like channels have been very difficult to obtain. Jiang et al. figured that part of the problem might be that the voltage sensor flapped around too much and prevented crystallization of the protein. Indeed, they found that if they pinned down the sensor domains by binding them to an antibody Fab fragment, they could obtain crystals of KvAP, a bacterial channel that is similar to mammalian voltage-gated K + channels. Structural analysis revealed that the voltage sensor appears to be a hydrophobic helix-turn-helix structure that the authors call a voltage-sensor paddle. If one assumes that the crystals accurately represent the conformation of the functional receptor in a membrane, the paddle appears to sit near the intracellular surface of the membrane. Although previous models suggested that the sensor might move within the proteinaceous channel complex of four subunits, the new structure indicates that the paddle instead likely moves through the membrane itself on the channel perimeter. This may explain pharmacological data showing that small lipid-soluble molecules easily access the voltage sensor. On the basis of the structure, the authors tagged key positions in the voltage sensor with biotinylated cysteine residues and monitored the presence of the sensor on the inner or outer surface of the membrane by measuring avidin binding. These experiments confirmed that the paddles flop back and forth through the membrane in response to voltage changes, acting as what the authors call hydrophobic cations attached to levers. Sigworth provides commentary on these results. Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B. T. Chait, R. Mackinnon, X-ray structure of a voltage-dependent K + channel. Nature 432 , 33-41 (2003). [Online Journal] Y. Jiang, V. Ruta, J. Chen, A. Lee, R. Mackinnon, The principle of gating charge movement in a voltage-dependent K + channel. Nature 432 , 42-48 (2003). [Online Journal] F. J. Sigworth, Life's transistors. Nature 432 , 21-22 (2003). [Online Journal]

Extending the Structure of an ABC Transporter to Atomic Resolution: Modeling and Simulation Studies of MsbA

Molecular modeling and simulation approaches have been use to generate a complete model of the prokaryotic ABC transporter MsbA from Escherichia coli, starting from the low-resolution structure-based Calpha trace (PDB code 1JSQ). MsbA is of some biomedical interest as it is homologous to mammalian transporters such as P-glycoprotein and TAP. The quality of the MsbA model is assessed using a combination of molecular dynamics simulations and static structural analysis. These results suggest that the approach adopted for MsbA may be of general utility for generating all atom models from low-resolution crystal structures of membrane proteins. Molecular dynamics simulations of the MsbA model inserted in a fully solvated octane slab (a membrane mimetic environment) reveal that while the monomer is relatively stable, the dimer is unstable and undergoes significant conformational drift on a nanosecond time scale. This suggests that the MsbA crystal dimer may not correspond to the MsbA dimer in vivo. An alternative model of the dimer is discussed in the context of available experimental data.

A knowledge-based scale for amino acid membrane propensity.

In this article, a membrane-propensity scale for amino acids is derived using only two ingredients: (i) a set of transmembrane helices segments from membrane protein crystal structures and (ii) the request that each component of the set has a free energy lower than that of a typical soluble protein sequence of the same length. Although the most widely used hydropathy scales satisfy this request, we use an optimization procedure that allows for extraction of an optimal scale, which correlates equally well with those scales. We show that, if the choice of the sequence database is accurate, significant knowledge-based scales, which are robust with respect to changes in the learning set, can be easily derived. The obtained scales can be used for transmembrane helices prediction. The predictive power of one of these scales is tested on membrane proteins, soluble proteins, and signal peptides databases, finding that its performances is comparable with those of the hydropathy scales.

Influence of the Environment in the Conformation of α-Helices Studied by Protein Database Search and Molecular Dynamics Simulations

The influence of the solvent on the main-chain conformation ( ϕ and Ψ dihedral angles) of α-helices has been studied by complementary approaches. A first approach consisted in surveying crystal structures of both soluble and membrane proteins. The residues of analysis were further classified as exposed to either the water (polar solvent) or the lipid (apolar solvent) environment or buried to the core of the protein (intermediate polarity). The statistical results show that the more polar the environment, the lower the value of ϕ i and the higher the value of Ψ i are. The intrahelical hydrogen bond distance increases in water-exposed residues due to the additional hydrogen bond between the peptide carbonyl oxygen and the aqueous environment. A second approach involved nanosecond molecular dynamics simulations of poly-Ala α-helices in environments of different polarity: water to mimic hydrophilic environments that can form hydrogen bonds with the peptide carbonyl oxygen and methane to mimic hydrophobic environments without this hydrogen bond capabilities. These simulations reproduce similar effects in ϕ and Ψ angles and intrahelical hydrogen bond distance and angle as observed in the protein survey analysis. The magnitude of the intrahelical hydrogen bond in the methane environment is stronger than in the water environment, suggesting that α-helices in membrane-embedded proteins are less flexible than in soluble proteins. There is a remarkable coincidence between the ϕ and Ψ angles obtained in the analysis of residues exposed to the lipid in membrane proteins and the results from computer simulations in methane, which suggests that this simulation protocol properly mimic the lipidic cell membrane and reproduce several structural characteristics of membrane-embedded proteins. Finally, we have compared the ϕ and Ψ torsional angles of Pro kinks in membrane protein crystal structures and in computer simulations.

Structures of membrane proteins determined at atomic resolution.

Following determination of the first crystal structure of the reaction center of Rhodopseudomonas viridis, a membrane protein, by X-ray crystal structure analysis at 3.0 A resolution, 18 X-ray crystal structures and two electron crystal structures of membrane proteins have been obtained at higher than 3.5 A resolution. Besides these integral membrane protein structures, three crystal structures of water-soluble proteins, which can enter membranes, have been determined by X-ray crystallography at high resolution. The structural features of membrane proteins have been summarized by inspecting these crystal structures. The polypeptide chain crosses the membrane in a helical conformation or a beta-strand. The central +10 A region of the transmembrane alpha-helix is dominated by hydrophobic residues. On both sides of the central region are concentrated polar aromatic residues. Charged residues are dominant around +15 A to +20 A. All the transmembrane beta-structures are found in pore-forming proteins. The central region of the transmembrane beta-structure is amphipathic with hydrophobic residues on the membrane exposed side. The distribution of amino acid residues on the membrane exposed surface of the transmembrane beta-structure is similar to that of the transmembrane alpha-helix. alpha-Helices anchoring the membrane surface region are amphipathic with hydrophobic residues inside and hydrophilic residues outside.