Accessibility of introduced cysteines in chemoreceptor transmembrane helices reveals boundaries interior to bracketing charged residues

Two hydrophobic sequences, 24 and 30 residues long, identify the membrane-spanning segments of chemoreceptor Trg from Escherichia coli. As in other related chemoreceptors, these helical sequences are longer than the minimum necessary for an alpha-helix to span the hydrocarbon region of a biological membrane. Thus, the specific positioning of the segments relative to the hydrophobic part of the membrane cannot be deduced from sequence alone. With the aim of defining the positioning for Trg experimentally, we determined accessibility of a hydrophilic sulfhydryl reagent to cysteines introduced at each position within and immediately outside the two hydrophobic sequences. For both sequences, there was a specific region of uniformly low accessibility, bracketed by regions of substantial accessibility. The two low-accessibility regions were each 19 residues long and were in register in the three-dimensional organization of the transmembrane domain deduced from independent data. None of the four hydrophobic-hydrophilic boundaries for these two membrane-embedded sequences occurred at a charged residue. Instead, they were displaced one to seven residues internal to the charged side chains bracketing the extended hydrophobic sequences. Many hydrophobic sequences, known or predicted to be membrane-spanning, are longer than the minimum necessary helical length, but precise membrane boundaries are known for very few. The cysteine-accessibility approach provides an experimental strategy for determining those boundaries that could be widely applicable.     

Complementary analysis of the vegetative membrane proteome of the human pathogen Staphylococcus aureus

The Gram-positive bacterium Staphylococcus aureus is a serious human pathogen causing a wide variety of diseases, and its increasing resistance toward all available antibiotics makes its further investigation absolutely essential. We examined the membrane proteome of exponentially growing cells of S. aureus COL because this subproteome plays a major role in the virulence of the bacterium in its host. In general, an analysis of membrane proteins is impeded by their hydrophobic nature as well as by a high abundance of many cytosolic proteins. The implementation of three different technologies, one-dimensional gel-LC, two-dimensional LC, and a membrane shaving approach combined with MS/MS analyses, enabled an identification of 271 integral and 86 peripheral membrane proteins from exponentially growing cells. In particular, the latter approach that combined membrane shaving with a subsequent chymotrypsin digest of integral membrane domains of proteins greatly facilitated the detection of hydrophobic peptides derived from membrane-spanning segments (713 peptides, 60% of all peptides) and therefore yielded almost exclusively highly hydrophobic integral membrane proteins (96.7%). A comparison of the various methods disclosed the one-dimensional gel-LC and the shaving approach to be highly complementary techniques. A combination of them will reveal a most comprehensive view on membrane proteomes.     

Forces involved in the assembly and stabilization of membrane proteins.

Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membrane- and water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the "positive-inside" rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning alpha-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chloroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin.     

Flexible regions within the membrane-embedded portions of polytopic membrane proteins

The conventional view of the structure of the membrane-embedded regions of integral membrane proteins is that they are in contact with lipids that interact with the hydrophobic surfaces of the polypeptide, and therefore have intrinsically rigid alpha-helical structures. Here, we briefly review the evidence that in the case of integral membrane proteins with many membrane spans (including membrane transporters and channels), some membrane peptide segments are more or less completely shielded from the lipid bilayer by other membrane polypeptide portions. These portions do not need to have alpha-helical structures and are likely to be much more flexible than typical membrane-spanning helices. The ability of the band 3 anion exchanger to accommodate anionic substrates of different sizes, geometries, and charge distributions suggests the presence of flexible regions in the active center of this protein. These flexible substructures may have important functional roles in membrane proteins, particularly in the mechanisms of membrane transporters and channels.     

Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits

AAA proteases are membrane-bound ATP-dependent proteases that are present in eubacteria, mitochondria and chloroplasts and that can degrade membrane proteins. Recent evidence suggests dislocation of membrane-embedded substrates for proteolysis to occur in a hydrophilic environment; however, next to nothing is known about the mechanism of this process. Here, we have analysed the role of the membrane-spanning domains of Yta10 and Yta12, which are conserved subunits of the hetero-oligomeric m-AAA protease in the mitochondria of Saccharomyces cerevisiae. We demonstrate that the m-AAA protease retains proteolytic activity after deletion of the transmembrane segments of either Yta10 or Yta12. Although the mutant m-AAA protease is still capable of processing cytochrome c peroxidase and degrading a peripheral membrane protein, proteolysis of integral membrane proteins is impaired. We therefore propose that transmembrane segments of m-AAA protease subunits have a direct role in the dislocation of membrane-embedded substrates.     

Amino acid composition of the membrane and aqueous domains of integral membrane proteins

To identify residues which might impart transport capability to the intramembranous regions of transport proteins, we surveyed available data for the 9991 amino acids contained in the aqueous and intramembranous regions of 24 integral membrane proteins: 10 transport (T) proteins and 14 nontransport (NT) proteins. Statistical comparison of percentage occurrence of each amino acid within T and NT samples provided a measure of "typical" composition of T and NT membrane-spanning regions, and showed that the residues partition into membrane and aqueous domains largely in accord with expectation from hydropathy indices. Comparison of aqueous and membrane domain composition between protein categories revealed a statistically similar distribution of residues in aqueous domains, but significant differences in membrane domains: seven residues (Asn, Asp, Gln, Glu, Phe, Pro, Tyr) were preferred in membrane regions of T proteins, and one (Val) was selectively excluded. Chemical and structural considerations suggested that three of these residues--Asn, Tyr, and Pro--are the most likely functional participants in transport processes.     

Genome-wide analysis of integral membrane proteins from eubacterial,archaean, and eukaryotic organisms.

We have carried out detailed statistical analyses of integral membrane proteins of the helix-bundle class from eubacterial, archaean, and eukaryotic organisms for which genome-wide sequence data are available. Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones. Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni-cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments. In all organisms, there is a tendency that membrane proteins either have many transmembrane segments with short connecting loops or few transmembrane segments with large extra-membraneous domains. Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii, follow the so-called "positive-inside" rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra-cytoplasmic segments.     

Hydrophobicity and prediction of the secondary structure of membrane proteins and peptides.

Reliability of the hydropathy method to predict the formation of membrane-spanning alpha-helices by integral membrane proteins and peptides whose structure is known from X-ray crystallography is analysed. It is shown that Kyte-Doolittle hydropathy plots do not predict accurately 22 transmembrane alpha-helices in the reaction centres (RC) of the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides (R-26). The accuracy of prediction for these proteins was improved using an optimised Kyte-Doolittle hydrophobicity scale. However, this hydrophobicity scale did not improve the predictions for the alphabeta-peptides of the B800-850 (LH2) complexes of the photosynthetic bacteria Rhodopseudomonas acidophila and Rhodospirillum molischianum, which were excluded from the optimisation procedure. The best and worst predictions of membrane-spanning alpha-helices for the RC proteins and LH2 peptides, respectively, were obtained with a propensity scale (PRC) calculated from the amino acid sequences and X-ray data for the RC proteins. A propensity scale (PLH) obtained using the amino acid sequences and X-ray data for the alphabeta-peptides of the LH2 complexes did not give an acceptable prediction of the transmembrane segments in the LH2 peptides; moreover, it markedly contradicted the PRC scale. Amino acids have been concluded to have no significant preference to localisation in transmembrane segments. Therefore, the predictive ability of the hydropathy methodology appears to be limited: the number of transmembrane segments can be correctly calculated for the best case only, and the lengths and positions of membrane-spanning alpha-helices in a protein amino acid sequence can not be predicted exactly.     

Membrane-associated tyrosine kinases as molecular switches

Tyrosine kinases can be associated with membranes as membrane-spanning integral membrane proteins or as intracellular peripheral membrane proteins. Both categories of tyrosine kinase transduce extracellular signals to the cytosol by switching between inactive and active states. Switching is achieved by changes in phosphorylation state and intra- and inter-molecular binding interactions. In turn, activated tyrosine kinases affect their substrates by changing their phosphorylation state and by binding them.     

Topogenic signals in integral membrane proteins

Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.     

Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.     

A small hydrophobic domain anchors leader peptidase to the cytoplasmic membrane of Escherichia coli

Leader peptidase is an enzyme of the Escherichia coli cytoplasmic membrane which removes amino-terminal leader sequences from many secreted and membrane proteins. Three potential membrane-spanning segments exist in the first 98 amino acids of leader peptidase. We have characterized the topology of leader peptidase based on its sensitivity to protease digestion. Proteinase K and trypsin treatment of right-side-out inner membrane vesicles and spheroplasts yields protected fragments of approximately 80 and 105 amino acid residues, respectively. We have shown that both fragments are derived from the amino terminus of the protein and that the smaller protected peptide can be derived from the larger. Removal of the third potential membrane-spanning segment (residues 82-98) does not affect the size of the proteinase K-protected fragment but does reduce the size of the trypsin-protected peptide. Because the proteinase K-protected fragment is about 9000 daltons, is derived from the amino terminus of leader peptidase, and its size is not affected when amino acids 82-98 are removed from the protein, it must extend from the amino terminus to approximately residue 80. Likewise, the trypsin-protected fragment must extend from the amino terminus to about residue 105. These data suggest a model for the orientation of leader peptidase in which the second hydrophobic stretch (residues 62-76) spans the cytoplasmic membrane and the third hydrophobic stretch resides in the periplasmic space.     

OPM: orientations of proteins in membranes database

SUMMARY: The Orientations of Proteins in Membranes (OPM) database provides a collection of transmembrane, monotopic and peripheral proteins from the Protein Data Bank whose spatial arrangements in the lipid bilayer have been calculated theoretically and compared with experimental data. The database allows analysis, sorting and searching of membrane proteins based on their structural classification, species, destination membrane, numbers of transmembrane segments and subunits, numbers of secondary structures and the calculated hydrophobic thickness or tilt angle with respect to the bilayer normal. All coordinate files with the calculated membrane boundaries are available for downloading. AVAILABILITY: http://opm.phar.umich.edu.     

The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane.

We report here, for the first time, the primary structure of uncoupling protein as established by amino acid sequencing. Like the ADP/ATP carrier, this protein has a tripartite structure comprising three similar sequences of approximately 100 residues each. These six 'repeats' exhibit striking conservation of several residues, in particular glycine and proline, at possible structurally strategic positions. Although the two proteins differ strongly in their amino acid composition, their sequences are distantly homologous. Three membrane-spanning alpha-helices can be deduced from hydropathy plots. A modified plot accounting for amphiphilic helices indicates 5-6 such alpha-segments. In addition an amphiphilic beta-strand of membrane-spanning length can be discerned. The tripartite sequence structure is also distinctly reflected in the hydropathy distribution. Based on the membrane disposition of the segments of the ADP/ATP carrier, a model for the transmembrane folding path of the polypeptide chain of the uncoupling protein is proposed.     

Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli.

The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix.     

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/.     

Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER

The integral endoplasmic reticulum (ER) membrane protein Shr3p is required for proper plasma membrane localization of amino acid permeases (AAPs) in yeast. In the absence of Shr3p AAPs are uniquely retained in the ER with each of their twelve membrane-spanning segments correctly inserted in the membrane. Here, we show that the membrane domain of Shr3p specifically prevents AAPs from aggregating, and thus, plays a critical role in assisting AAPs to fold and correctly attain tertiary structures required for ER exit. Also, we show that the integral ER proteins, Gsf2p, Pho86p, and Chs7p, function similarly to Shr3p. In cells individually lacking one of these components only their cognate substrates, hexose transporters, phosphate transporters, and chitin synthase-III, respectively, aggregate and consequently fail to exit the ER membrane. These findings indicate that polytopic membrane proteins depend on specialized membrane-localized chaperones to prevent inappropriate interactions between membrane-spanning segments as they insert and fold in the lipid bilayer of the ER membrane.     

Electrostatic destabilization of the cytochrome b6f complex in the thylakoid membrane.

Three of the membrane-spanning polypeptides of the chloroplast cytochrome (cyt) b6f complex were sequentially released from the thylakoid membrane, in the order cyt b6, suIV and Rieske iron-sulfur protein, as the pH was increased from 10 to 12, a protocol usually employed to remove peripheral proteins from membranes. The fourth polypeptide of the cyt b6f complex, cyt f, which spans the membrane once, was apparently not released. The pH values for half-release at low ionic strength were approximately 10.7, 11.1 and 11.3 respectively. The separation of the polypeptides of the complex and the sequential release is readily seen at pH 11, where the loss from the membrane of cyt b6, suIV and Fe iron-sulfur center is approximately 90%, 50% and 20%, respectively. the release of cyt b6 from the membrane was reflected by the absence of its characteristic reduced minus oxidized absorbance signal. The pH values at which the release occurred increased as the ionic strength was raised, implying that the release of the b6f polypeptides arises from extrusion due to repulsive electrostatic interactions probably caused by deprotonation of tyrosine and lysine residues. The lipid content of the released polypeptides was very low, consistent with the observation of a non-membranous state. It is proposed that the pH-dependent extrusion requires two electrostatic effects at alkaline pH higher than approximately 10.5: (i) increased electrostatic repulsion between neighbouring polypeptides of the complex, arising from increased net negative charge in the peripheral segments of these polypeptides, which can cause separation of the polypeptides from the complex; and (ii) ionization of residues such as tyrosine in the membrane-spanning alpha-helices, and neutralization of residues such as lysine which can bind to the negative membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.     

Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion

Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.