Topology Of Integral Membrane Proteins Research Articles

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).

Direct Simulation of Early-Stage Sec-Facilitated Protein Translocation

Direct simulations reveal key mechanistic features of early-stage protein translocation and membrane integration via the Sec-translocon channel. We present a novel computational protocol that combines non-equilibrium growth of the nascent protein with microsecond timescale molecular dynamics trajectories. Analysis of multiple, long timescale simulations elucidates molecular features of protein insertion into the translocon, including signal-peptide docking at the translocon lateral gate (LG), large lengthscale conformational rearrangement of the translocon LG helices, and partial membrane integration of hydrophobic nascent-protein sequences. Furthermore, the simulations demonstrate the role of specific molecular interactions in the regulation of protein secretion, membrane integration, and integral membrane protein topology. Salt-bridge contacts between the nascent-protein N-terminus, cytosolic translocon residues, and phospholipid head groups are shown to favor conformations of the nascent protein upon early-stage insertion that are consistent with the Type II (N(cyt)/C(exo)) integral membrane protein topology, and extended hydrophobic contacts between the nascent protein and the membrane lipid bilayer are shown to stabilize configurations that are consistent with the Type III (N(exo)/C(cyt)) topology. These results provide a detailed, mechanistic basis for understanding experimentally observed correlations between integral membrane protein topology, translocon mutagenesis, and nascent-protein sequence.

The membrane integration of a naturally occurring α-helical hairpin

Helical hairpins, two closely spaced helical membrane spanning segments separated by a short surface turn, are thought to be common in integral membrane proteins. Here, we study the membrane integration of a naturally occurring helical hairpin from the secretory Na +–K +–2Cl − cotransporter NKCC1. This sequence is only slightly longer and significantly less hydrophobic than a previously identified minimal poly-leucine model hairpin structure. Using site directed mutagenesis we document the importance of the turn propensity of the amino acids in the intervening surface turn but, somewhat surprisingly, our results indicate that the formation of this natural hairpin apparently does not depend on specific helix–helix interactions. Our results suggest that helical hairpins may be formed quite readily from even minimally hydrophobic sequences separated by a short, sufficiently strong, turn signal, and that current methods for predicting integral membrane protein topology may miss many similar short helical hairpin sequences. Thus the occurrence of these structures may be much more common than presently thought.

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.

Membrane protein topology: phospholipids call the shots

The insertion and assembly of integral membrane proteins into biological membranes is an important and largely uncharacterized process in many organisms. Studies using Escherichia coli have elucidated the roles of certain amino acids in determining the topology of integral membrane proteins. However, the role of the environment in which the protein sits – the phospholipid bilayer – has been largely ignored in these studies. Now, William Dowhan and his group in Houston, USA, have made some exciting observations relating membrane lipid composition to the topology of a well-studied integral membrane protein [ 1. Bogdanov M. et al. A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition. EMBO J. 2002; 21: 2107-2116 Crossref PubMed Scopus (190) Google Scholar ].

20] Analysis of transporter topology using deletion and epitope tagging

Publisher Summary This chapter describes the analysis of transporter topology using deletion and epitope tagging. Several strategies have been employed to elucidate the structure and transmembrane topology of transport proteins. An understanding of transporter structure is crucial for determining the correlation between transporter structure and function. In addition, structural knowledge aids in the design of more specific transporter reagents, which may prove therapeutically useful. Current studies of the transmembrane topology of integral membrane proteins using deletion analysis and epitope tagging derive from gene fusion methods designed to study secretion and topology of bacterial proteins. In these original experiments, a modified form of the Escherichia coli phoA gene, which encodes the secreted periplasmic enzyme alkaline phosphatase, was fused to the C-terminal portion of a protein of interest. Therefore, the level of alkaline phosphatase activity is a direct indication of the location of the enzyme in bacteria and a useful tool for discerning the secretion or topogenic properties of bacterial proteins. The phoA gene fusion method has been used to determine the transmembrane topology of the C-terminal half of the bacterial glutamate transporter in E. coli .

Prl Mutations in the Escherichia coli secG Gene

SecG, an integral membrane component of the Escherichia coli preprotein translocase, contributes to the efficiency of the export process by undergoing cycles of topology inversion in the membrane, coupled with the insertion-deinsertion cycles of SecA. We have previously identified sec alleles of secG that cause a generalized secretion defect. In this study, by screening mutagenized secG libraries for suppressors of a malE signal sequence mutation, we isolated prl alleles of secG. By analogy with secY/prlA, secA/prlD, and secE/prlG, secG could therefore be called secG/prlH. The prlH mutations affect 13 codons distributed along the secG sequence, and none map to the codons affected by sec mutations. prlH suppressors suppress a variety of signal sequence mutations and they allow export of alkaline phosphatase lacking its entire signal sequence. Although secG was not identified in previous selections for prl mutants, several prlH alleles are as strong as the strongest known prlG alleles of secE. Some prlH alleles can also promote the export of alkaline phosphatase fused to predicted cytoplasmic domains of UhpT, an integral membrane protein. These results support the notion that SecG contributes to signal sequence recognition, and suggest that it may also contribute to the topology of integral membrane proteins.

A Model Recognition Approach to the Prediction of All-Helical Membrane Protein Structure and Topology

This paper describes a new method for the prediction of the secondary structure and topology of integral membrane proteins based on the recognition of topological models. The method employs a set of statistical tables (log likelihoods) complied from well-characterized membrane protein data, and a novel dynamic programming algorithm to recognize membrane topology models by expectation maximization. The statistical tables show definite biases toward certain amino acid species on the inside, middle, and outside of a cellular membrane. Using a set of 83 integral membrane protein sequences taken from a variety of bacterial, plant, and animal species, and a strict jackknifing procedure, where each protein (along with any detectable homologues) is removed from the training set used to calculate the tables before prediction, the method successfully predicted 64 of the 83 topologies, and of the 37 complex multispanning topologies 34 were predicted correctly.

Reporter epitopes: a novel approach to examine transmembrane topology of integral membrane proteins applied to the alpha 1 subunit of the nicotinic acetylcholine receptor.

The development of a novel immunological method called the "reporter epitope" technique to probe the transmembrane topology of integral membrane proteins is described. Using this method, synthetic oligonucleotides encoding epitopes (reporter epitopes) for well characterized monoclonal antibodies (reporter mAbs) were inserted at various locations within the human acetylcholine receptor (AChR) alpha 1 subunit cDNA. The engineered subunits were then expressed along with Torpedo beta 1, gamma, and delta subunits in Xenopus oocytes, and the transmembrane location of the site of insertion was determined by the binding of the 125I-labeled reporter mAbs to whole oocytes. Control reporter epitope insertions at alpha 347 exhibited the expected cytoplasmic location. Reporter epitopes inserted at alpha 429 are located on the extracellular surface. Reporter epitopes that are 16-48 amino acids long do not disrupt assembly or function of hybrid AChRs when inserted near the carboxy terminus (at alpha 429) or in the large cytoplasmic domain (at alpha 347). However, because two reporter epitopes inserted at alpha 157 obliterated subunit assembly and a third reporter epitope when tolerated at this position was inaccessible from the extracellular surface and only marginally accessible after detergent solubilization of the AChRs, a definitive transmembrane location for this region was not possible. Nonetheless, the use of this approach has been successfully demonstrated, and it may be generally applicable to the study of other integral membrane proteins.

Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues.

Positively charged amino acids have been shown to be important elements in targeting-peptides that direct proteins into mitochondria, nuclei, and the secretory pathways of both prokaryotic and eukaryotic cells. The 'positive-inside' rule, which observes that regions of polytopic (multi-spanning) membrane proteins facing the cytoplasm are generally enriched in arginyl and lysyl residues whereas translocated regions are largely devoid of these residues, implies that the distribution of positively charged amino acids may also be a major determinant of the transmembrane topology of integral membrane proteins. If this is indeed the case, it should be possible to predictably alter the topology of a polytopic protein by site-directed insertions and/or deletions of positively charged residues in critical locations. I now describe a derivative of Escherichia coli leader peptidase, a polytopic inner-membrane protein, that switches from sec-gene-dependent membrane insertion with a Nout-Cout transmembrane topology to sec-gene-independent insertion with a Nin-Cin topology in response to the addition of four positively charged lysines to its N terminus.