High-resolution Structures Of Membrane Proteins Research Articles

Computational Studies on the Interactions of Inhalational Anesthetics with Proteins

Despite the widespread clinical use of anesthetics since the 19th century, a clear understanding of the mechanism of anesthetic action has yet to emerge. On the basis of early experiments by Meyer, Overton, and subsequent researchers, the cell's lipid membrane was generally concluded to be the primary site of action of anesthetics. However, later experiments with lipid-free globular proteins, such as luciferase and apoferritin, shifted the focus of anesthetic action to proteins. Recent experimental studies, such as photoaffinity labeling and mutagenesis on membrane proteins, have suggested specific binding sites for anesthetic molecules, further strengthening the proteocentric view of anesthetic mechanism. With the increased availability of high-resolution crystal structures of ion channels and other integral membrane proteins, as well as the availability of powerful computers, the structure-function relationship of anesthetic-protein interactions can now be investigated in atomic detail. In this Account, we review recent experiments and related computer simulation studies involving interactions of inhalational anesthetics and proteins, with a particular focus on membrane proteins. Globular proteins have long been used as models for understanding the role of protein-anesthetic interactions and are accordingly examined in this Account. Using selected examples of membrane proteins, such as nicotinic acetyl choline receptor (nAChR) and potassium channels, we address the issues of anesthetic binding pockets in proteins, the role of conformation in anesthetic effects, and the modulation of local as well as global dynamics of proteins by inhaled anesthetics. In the case of nicotinic receptors, inhalational anesthetic halothane binds to the hydrophobic cavity close to the M2-M3 loop. This binding modulates the dynamics of the M2-M3 loop, which is implicated in allosterically transmitting the effects to the channel gate, thus altering the function of the protein. In potassium channels, anesthetic molecules preferentially potentiate the open conformation by quenching the motion of the aromatic residues implicated in the gating of the channel. These simulations suggest that low-affinity drugs (such as inhalational anesthetics) modulate the protein function by influencing local as well as global dynamics of proteins. Because of intrinsic experimental limitations, computational approaches represent an important avenue for exploring the mode of action of anesthetics. Molecular dynamics simulations-a computational technique frequently used in the general study of proteins-offer particular insight in the study of the interaction of inhalational anesthetics with membrane proteins.

Prediction of membrane protein structures with complex topologies using limited constraints

Reliable structure-prediction methods for membrane proteins are important because the experimental determination of high-resolution membrane protein structures remains very difficult, especially for eukaryotic proteins. However, membrane proteins are typically longer than 200 aa and represent a formidable challenge for structure prediction. We have developed a method for predicting the structures of large membrane proteins by constraining helix-helix packing arrangements at particular positions predicted from sequence or identified by experiments. We tested the method on 12 membrane proteins of diverse topologies and functions with lengths ranging between 190 and 300 residues. Enforcing a single constraint during the folding simulations enriched the population of near-native models for 9 proteins. In 4 of the cases in which the constraint was predicted from the sequence, 1 of the 5 lowest energy models was superimposable within 4 A on the native structure. Near-native structures could also be selected for heme-binding and pore-forming domains from simulations in which pairs of conserved histidine-chelating hemes and one experimentally determined salt bridge were constrained, respectively. These results suggest that models within 4 A of the native structure can be achieved for complex membrane proteins if even limited information on residue-residue interactions can be obtained from protein structure databases or experiments.

Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination.

Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This "discovery-oriented" pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column--a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker's yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes.

β-Barrel Proteins That Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane beta-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.

Inter-residue interactions in protein structures exhibit power-law behavior

Inter-residue interactions play an essential role in driving protein folding, and analysis of these interactions increases our understanding of protein folding and stability and facilitates the development of tools for protein structure and function prediction. In this work, we systematically characterized the change of inter-residue interactions at various sequence separation cutoffs using two protein datasets. The first set included 100 diverse, nonredundant and high-resolution soluble protein structures, covering all four major structural classes, all-alpha, alpha/beta, alpha+beta, and all-beta; and the second set included 20 diverse, nonredundant and high-resolution membrane protein structures, representing 19 unique superfamilies. It was shown that the average number of inter-residue interactions in structures of both datasets displays the power-law behavior. Fitting parameters of the power-law function are directly related to the structural classes analyzed. These findings provided further insight into the distribution of short-, medium-, and long-range inter-residue interactions in both soluble and membrane proteins and could be used for protein structure prediction.

A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization

A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.

A dynamic view of peptides and proteins in membranes.

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins.

High-Resolution 3D Structure Determination of Kaliotoxin by Solid-State NMR Spectroscopy

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

Surface recognition elements of membrane protein oligomerization

Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts.

Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction

De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction.

How strongly do sequence conservation patterns and empirical scales correlate with exposure patterns of transmembrane helices of membrane proteins?

Given the difficulty in determining high-resolution structures of helical membrane proteins, sequence-based prediction methods can be useful in elucidating diverse physiological processes mediated by this important class of proteins. Predicting the angular orientations of transmembrane (TM) helices about the helix axes, based on the helix parameters from electron microscopy data, is a classical problem in this regard. This problem has triggered the development of a number of different empirical scales. Recently, sequence conservation patterns were also made use of for improved predictions. Empirical scales and sequence conservation patterns (collectively termed as "prediction scales") have also found frequent applications in other research areas of membrane proteins: for example, in structure modeling and in prediction of buried TM helices. This trend is expected to grow in the near future unless there are revolutionary developments in the experimental characterization of membrane proteins. Thus, it is timely and imperative to carry out a comprehensive benchmark test over the prediction scales proposed so far to determine their pros and cons. In the current analysis, we use exposure patterns of TM helices as a golden standard, because if one develops a prediction scale that correlates perfectly with exposure patterns of TM helices, it will enable one to predict buried residues (or buried faces) of TM helices with an accuracy of 100%. Our analysis reveals several important points. (1) It demonstrates that sequence conservation patterns are much more strongly correlated with exposure patterns of TM helices than empirical scales. (2) Scales that were specifically parameterized using structure data (structure-based scales) display stronger correlation than hydrophobicity-based scales, as expected. (3) A nonnegligible difference is observed among the structure-based scales in their correlational property, suggesting that not every learning algorithm is equally effective. (4) A straightforward framework of optimally combining sequence conservation patterns and empirical scales is proposed, which reveals that improvements gained from combining the two sources of information are not dramatic in almost all cases. In turn, this calls for the development of fundamentally different scales that capture the essentials of membrane protein folding for substantial improvements.

Structural Genomics: The Ultimate Approach for Rational Drug Design

Structural genomics can be defined as structural biology on a large number of target proteins in parallel. This approach plays an important role in modern structure-based drug design. Although a number of structural genomics initiatives have been initiated, relatively few are associated with integral membrane proteins. This indicates the difficulties in expression, purification, and crystallization of membrane proteins, which has also been confirmed by the existence of some 100 high-resolution structures of membrane proteins among the more than 30,000 entries in public databases. Paradoxically, membrane proteins represent 60-70% of current drug targets and structural knowledge could both improve and speed up the drug discovery process. In order to improve the success rate for structure resolution of membrane proteins structural genomics networks have been established.

Transmembrane helix prediction: a comparative evaluation and analysis

The prediction of transmembrane (TM) helices plays an important role in the study of membrane proteins, given the relatively small number (approximately 0.5% of the PDB) of high-resolution structures for such proteins. We used two datasets (one redundant and one non-redundant) of high-resolution structures of membrane proteins to evaluate and analyse TM helix prediction. The redundant (non-redundant) dataset contains structure of 434 (268) TM helices, from 112 (73) polypeptide chains. Of the 434 helices in the dataset, 20 may be classified as 'half-TM' as they are too short to span a lipid bilayer. We compared 13 TM helix prediction methods, evaluating each method using per segment, per residue and termini scores. Four methods consistently performed well: SPLIT4, TMHMM2, HMMTOP2 and TMAP. However, even the best methods were in error by, on average, about two turns of helix at the TM helix termini. The best and worst case predictions for individual proteins were analysed. In particular, the performance of the various methods and of a consensus prediction method, were compared for a number of proteins (e.g. SecY, ClC, KvAP) containing half-TM helices. The difficulties of predicting half-TM helices suggests that current prediction methods successfully embody the two-state model of membrane protein folding, but do not accommodate a third stage in which, e.g., short helices and re-entrant loops fold within a bundle of stable TM helices.

The lipid/protein interface as xenobiotic target site

High-resolution X-ray diffraction structures of integral membrane proteins have revealed various binding modes of lipids, but current spectroscopic studies still use uniform macroscopic binding constants to describe lipid binding. The Adair approach employing microscopic lipid-binding constants has previously been taken to explain the enhancement of agonist binding to the nicotinic acetylcholine receptor by general anaesthetics in terms of the competitive displacement of essential lipid activator molecules [Walcher S, Altschuh J & Sandermann H (2001) J. Biol. Chem.276, 42191-42195]. This approach was extended to tadpole narcosis induced by alcohols. A single class, or two different classes of lipid activator binding sites, are considered. Microscopic lipid and inhibitor binding constants are derived and allow a close fit to dose-response curves of tadpole narcosis on the basis of a preferential displacement of more loosely bound essential lipid activator molecules. This study illustrates the potential of the Adair approach to resolve protein-bound lipid populations.

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.

Crystallization of Transmembrane Proteins in cubo: Mechanisms of Crystal Growth and Defect Formation

Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.

Crystallography of membrane proteins, major targets in drug design.

Protein crystallography has the potential to accelerate drug discovery greatly. High-resolution structures of membrane proteins of pharmaceutical interest open new perspectives in drug design. Recent structural data obtained for cyclooxygenases, monoamine oxidase, squalene cyclase, rhodopsine, porins, aquaporins, and ABC transporters are presented and briefly discussed.

Approaches to determining membrane protein structures to high resolution: do selections of subpopulations occur?

Three different methods are currently used for the study of high-resolution structures of membrane proteins: X-ray crystallography, electron crystallography, and nuclear magnetic resonance (NMR) spectroscopy. Thus far, all methods combined have yielded a rather modest number of crystal structures that have been solved at the atomic level. It is hypothesized here that different methods may select different populations of proteins on the basis of various properties. Thus, protein stability may be a significant factor in the formation of three-dimensional (3D) crystals from detergent solutions, since exposure of hydrophobic protein zones to water may cause structural perturbation or denaturation in conformationally labile proteins. This is different in the formation of two-dimensional (2D) crystals where a protein remains protected in its native membrane environment. A biological selection mechanism may therefore be operative in that highly ordered lattices may form only if strong protein–protein interactions are relevant in vivo, thereby limiting the number of proteins that are amenable to electron crystallography. Keeping a protein in a bilayer environment throughout 3D crystallization maintains the lateral pressure existing in native membranes. This can be accomplished by using lipidic cubic phases. Alternatively, the hydrophobic interface of a membrane protein may be spared from contact with water by crystallization from organic solvents where the polar caps are protected in reverse micelles by using appropriate detergents. Some of the criteria that are useful in optimizing the various approaches are given. While the usefulness of complementary methods seems obvious, the results presented may be particularly critical in recognizing key problems in other structural approaches.

K+ channels lacking the 'tetramerization' domain: implications for pore structure.

The difficulty of obtaining high-resolution structures of integral membrane proteins has been a frustrating barrier to understanding the membrane-based functions of living cells. The mere handful of such structures stands out in dismal contrast to the cornucopia of water-soluble proteins comprehensible at the atomic level. Nevertheless, crystallographically tractable preparations of aqueous domains of membrane proteins have provided molecular insight into phenomena as varied as chemotaxis, immune cell responses to antigens, viral infectivity and cellular synthesis of ATP. Recently, the first structural glimpse of a neuronal ion channel was reported - the T1, or 'tetramerization,' domain of a Shaker-type voltage-gated K+ channel at 1.6 A resolution. The isolated domain associates into a water-soluble four-fold symmetric homotetramer. This structure prompted the novel, provocative proposal that the T1 domain is an essential component of the ion permeation pathway, forming a previously unsuspected ion-coordinating constriction on the cytoplasmic side of the channel and acting as the receptor for the pore-blocking 'ball and chain' inactivation peptide. It has also been commonly conjectured that the T1 domain is required for tetramerization in the channel maturation process. By studying the detailed properties of Shaker K+ channels in which the T1 domain is deleted, we show all these proposals to be invalid. The structure of the T1 domain expressed in isolation is therefore unlikely to mirror in detail its structure when attached to the ion-conducting channel.

Characterization and modeling of membrane proteins using sequence analysis

The current libraries of amino acid sequences of membrane proteins are a valuable resource for the analysis of elements common to these proteins. Multiple-sequence alignment techniques and the identification of conserved features of transmembrane segments have improved the prediction of membrane protein topology. Molecular modeling in combination with structural studies or site-directed mutagenesis is proving to be a powerful link between theory and experiment. Unfortunately, the number of high-resolution structures of intrinsic membrane proteins, although increased recently, presents a restricted and perhaps biased view of membrane protein structure.