Membrane-protein topology

In the world of membrane proteins, topology defines an important halfway house between the amino-acid sequence and the fully folded three-dimensional structure. Although the concept of membrane-protein topology dates back at least 30 years, recent advances in the field of translocon-mediated membrane-protein assembly, proteome-wide studies of membrane-protein topology and an exponentially growing number of high-resolution membrane-protein structures have given us a deeper understanding of how topology is determined and of how it evolves.     

Normal-mode refinement of anisotropic thermal parameters for potassium channel KcsA at 3.2 A crystallographic resolution

We report a normal-mode method for anisotropic refinement of membrane-protein structures, based on a hypothesis that the global near-native-state disordering of membrane proteins in crystals follows low-frequency normal modes. Thus, a small set of modes is sufficient to represent the anisotropic thermal motions in X-ray crystallographic refinement. By applying the method to potassium channel KcsA at 3.2 A, we obtained a structural model with an improved fit with the diffraction data. Moreover, the improved electron density maps allowed for large structural adjustments for 12 residues in each subunit, including the rebuilding of 3 missing side chains. Overall, the anisotropic KcsA structure at 3.2 A was systematically closer to a 2.0 A KcsA structure, especially in the selectivity filter. Furthermore, the anisotropic thermal ellipsoids from the refinement revealed functionally relevant structural flexibility. We expect this method to be a valuable tool for structural refinement of many membrane proteins with moderate-resolution diffraction data.     

Helical packing patterns in membrane and soluble proteins

This article presents the results of a detailed analysis of helix-helix interactions in membrane and soluble proteins. A data set of interacting pairs of helices in membrane proteins of known structure was constructed and a structure alignment algorithm was used to identify pairs of helices in soluble proteins that superimpose well with pairs of helices in the membrane-protein data set. Most helix pairs in membrane proteins are found to have a significant number of structural homologs in soluble proteins, although in some cases, primarily involving irregular helices, no close homologs exist. An analysis of geometric relationships between interacting helices in the two sets of proteins identifies some differences in the distributions of helix length, interfacial area, packing angle, and distance between the polypeptide backbones. However, a subset of soluble-protein helix pairs that are close structural homologs to membrane-protein helix pairs exhibits distributions that mirror those observed in membrane proteins. The larger average interface size and smaller distance of closest approach seen for helices in membrane proteins appears due in part to a relative enrichment of alanines and glycines, particularly as components of the AxxxA and GxxxG motifs. It is argued that membrane helices are not on average more tightly packed than helices in soluble proteins; they are simply able to approach each other more closely. This enables them to interact over longer distances, which may in turn facilitate their remaining in contact over much of the width of the lipid bilayer. The close structural similarity seen between some pairs of helices in membrane and soluble proteins suggests that packing patterns observed in soluble proteins may be useful in the modeling of membrane proteins. Moreover, there do not appear to be fundamental differences between the magnitude of the forces that drive helix packing in membrane and soluble proteins, suggesting that strategies to make membrane proteins more soluble by mutating surface residues are likely to encounter success, at least in some cases.     

Emerging functional roles for the glycosyl-phosphatidylinositol membrane protein anchor

Conclusion Experimental evidence has accumulated over the past few years to suggest that the GPI protein anchor may play a broad role in the regulation of membrane protein function. The significant changes in the biophysical properties of proteins that are membrane-anchored through GPI in lieu of a hydrophobic transmembrane peptide indicates a variety phobic transmembrane peptide indicates a variety of potential new functions served by the anchor structure itself. Moreover, the number of structural variations within the family of GPI molecules indicates a further opportunity for subspecialization of such anchored proteins, especially regarding cellular localization, mobility, metabolism and susceptibility to enzymatically-induced release. It is likely that further exploration of the structure and function of the GPI anchor may reveal additional roles for this unusual mechanism of membrane-protein attachment.     

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios.     

Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.     

Biosynthesis and structural composition of gap junction intercellular membrane channels

Gap junction channels assemble as dodecameric complexes, in which a hexameric connexon (hemichannel) in one plasma membrane docks end-to-end with a connexon in the membrane of a closely apposed cell to provide direct cell-to-cell communication. Synthesis, assembly, and trafficking of the gap junction channel subunit proteins referred to as connexins, largely appear to follow the general secretory pathway for membrane proteins. The connexin subunits can assemble into homo-, as well as distinct hetero-oligomeric connexons. Assembly appears to be based on specific signals located within the connexin polypeptides. Plaque formation by the clustering of gap junction channels in the plane of the membrane, as well as channel degradation are poorly understood processes that are topics of current research. Recently, we tagged connexins with the autofluorescent reporter green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants and combined this reporter technology with single, and dual-color, high resolution deconvolution microscopy, computational volume rendering, and time-lapse microscopy to examine the detailed organization, structural composition, and dynamics of gap junctions in live cells. This technology provided for the first time a realistic, three-dimensional impression of gap junctions as they appear in the plasma membranes of adjoining cells, and revealed an excitingly detailed structural organization of gap junctions never seen before in live cells. Here, I summarize recent progress in areas encompassing the synthesis, assembly and structural composition of gap junctions with a special emphasis on the recent results we obtained using cell-free translation/ membrane-protein translocation, and autofluorescent reporters in combination with live-cell deconvolution microscopy.     

The complex architecture of oxygenic photosynthesis

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on earth. The primary step in this process - the conversion of sunlight into chemical energy - is driven by four, multisubunit, membrane-protein complexes that are known as photosystem I, photosystem II, cytochrome b(6)f and F-ATPase. Structural insights into these complexes are now providing a framework for the exploration not only of energy and electron transfer, but also of the evolutionary forces that shaped the photosynthetic apparatus.     

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.     

Palmitoylation-dependent protein sorting

S-palmitoylation is a posttranslational modification that regulates membrane-protein interactions. However, palmitate is more than just a hydrophobic membrane anchor, as many different types of protein are palmitoylated, including transmembrane proteins. Indeed, there is now compelling evidence that palmitoylation plays a key role in regulating various aspects of protein sorting within the cell.     

Membrane biochips

Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form H_II-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Gαq/11 protein and hence, signal transduction.     

AAA proteases: cellular machines for degrading membrane proteins

AAA proteases are a conserved class of ATP-dependent proteases that mediate the degradation of membrane proteins in bacteria, mitochondria and chloroplasts. They combine proteolytic and chaperone-like activities and thus form a membrane-integrated quality-control system. Inactivation of AAA proteases causes severe defects in various organisms, including neurodegeneration in humans. Proteolysis by AAA proteases is modulated by another membrane-protein complex that is composed of prohibitins in eukaryotic cells and related proteins in bacteria.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Mechanisms of membrane permeabilization by picornavirus 2B viroporin

Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane-protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.     

Membrane proteins and squalene-hydrosqualene profile in methanoarchaeon Methanothermobacter thermautotrophicus resistant to N,N′-dicyclohexylcarbodiimide

The biochemical basis of a defective bioenergetic system was attempted to be determined in N,N'-dicyclohexylcarbodiimide (DCCD)-resistant mutant of Methanothermobacter thermautotrophicus. Components participating in the maintenance of methanoarchaeal membrane structure and function, such as the composition of the mixture of squalene and its hydrosqualene derivatives and also properties of membrane-associated proteins were compared in wild-type and mutant cells. The impairment of the bioenergetic system in DCCD-resistant mutant was detectable in the membrane-protein profile; it was also accompanied by changes in proportions of squalene-hydrosqualenes.     

Identification and evolution of dual-topology membrane proteins

Integral membrane proteins are generally believed to have unique membrane topologies. However, it has been suggested that dual-topology proteins that adopt a mixture of two opposite orientations in the membrane may exist. Here we show that the membrane orientations of five dual-topology candidates identified in Escherichia coli are highly sensitive to changes in the distribution of positively charged residues, that genes in families containing dual-topology candidates occur in genomes either as pairs or as singletons and that gene pairs encode two oppositely oriented proteins whereas singletons encode dual-topology candidates. Our results provide strong support for the existence of dual-topology proteins and shed new light on the evolution of membrane-protein topology and structure.     

A procedure for membrane-protein reconstitution and the functional reconstitution of the anion transport system of the human-erythrocyte membrane.

A short procedure for the isolation of band-3 protein, the protein responsible for anion exchange in erythrocytes, in a reasonable degree of purity was developed. Using this protein preparation and a novel procedure for membrane-protein reconstitution, vesicles displaying the basic features of the anion-exchange system of the erythrocyte were obtained. The reconstitution procedure is based on slow direct removal of Triton X-100 from aqueous lipid/detergent solutions. According to the composition of the reconstitution medium, either small single-walled or large multi-walled vesicles are obtained. The procedure conserves protein properties well, as is revealed by the similarity of the rates of SO4(2-) exchange in erythrocytes and reconstituted vesicles when corrected for the relevant volumes. A number of functional features of the exchange system were studied and compared with those of the native membrane.