No abstract is provided for this article.
Membrane proteins destined for insertion into the inner membrane of E. coli are synthesized by ribosomes bound to the SecYEG translocon. During co‐translational membrane integration, transmembrane α‐helical segments in the nascent chain exit the translocon through a lateral gate that opens towards the surrounding membrane, but the mechanism of gate opening and lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when inside the translocon. Current work in our lab has shown that a force proportional to the free energy of membrane insertion is exerted on a transmembrane helix when it transits through the translocon channel, providing a direct insight into the dynamics of membrane integration.
No abstract is provided for this article.
As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physicochemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans‐membrane proteins.
The major aim of the studies that this thesis is based on has been to better define the topological determinants of the formation of so-called helical hairpins during membrane protein assembly in the ER membrane.The helical hairpin is a basic folding unit in membrane proteins. It is composed of two closely spaced transmembrane helices with a short connecting loop and it is believed to be inserted into the membrane as one compact unit. It is becoming increasingly clear that the helical hairpin is a very common structural element in membrane proteins and a detailed understanding of its properties is of central importance.We demonstrate that the efficiency of formation of helical hairpins depends both on the overall length of the hydrophobic segment, on the amino acids flanking the transmembrane segment, and on the identity of the central, potentially turn-forming residues. We also show that interhelical hydrogen bonds between pairs of Asn or Asp residues can induce helical hairpin formation.A detailed topology mapping is also reported for the Escherichia coli inner membrane chloride channel YadQ, a protein for which the X-ray structure is known. Our results provide a critical test of the reporter fusion approach and offer new insights into the YadQ folding pathway.In summary, the results present in this thesis have increased our understanding of the determinants of membrane protein topology and structure. Furthermore, the information obtained can be used to improve current models for predictions of membrane protein topology.
1. Introduction 286 2. Membrane protein assembly in E. coli 286 2.1. Role of the SRP 287 2.2. YidC – a translocon component devoted to membrane proteins? 287 2.3. The TAT pathway 288 2.4. ‘Spontaneous’ membrane protein insertion 288 3. Membrane protein assembly in the ER 289 3.1. How TM segments exit the translocon 289 3.2. Proteins with multiple topologies 290 3.3. Stop-transfer effector sequences 291 3.4. Non-hydrophobic TM segments? 291 3.5. ‘Frustrated’ topologies 291 3.6. N-tail translocation across the ER 292 4. Membrane protein assembly in mitochondria 292 4.1. The Oxa1p pathway 292 4.2. The TIM22/54 pathway 293 5. Evolution of membrane protein topology 293 5.1. RnfA/RnfE – two homologous proteins with opposite topologies 293 5.2. YrbG – duplicating an odd number of TMs 294 6. Genome-wide analysis of membrane proteins 295 6.1. Prediction methods 295 6.2. How many membrane proteins are there? 295 6.3. The positive-inside rule 296 6.4. Dominant classes of membrane proteins 296 7. The structure of transmembrane α-helices 296 7.1. What TM helices look like 297 7.2. The ‘helical hairpin’ 297 7.3. Prolines in TM helices 297 7.4. Charged residues in TM helices: the ‘snorkel’ effect 298 7.5. The ‘aromatic belt’ 298 8. Helix–helix packing in a membrane environment 298 8.1. Lessons learnt from glycophorin A 298 8.2. Genetic screens for helix–helix interactions 299 8.3. Statistical studies 299 8.4. Membrane protein folding 299 9. Recent 3D structures 300 9.1. KcsA – the first ion channel 300 9.2. MscL – sensing lateral pressure changes 300 9.3. The cytochrome bc 1 complex 300 9.4. Fumarate reductase 301 9.5. Bacteriorhodopsin – watching a membrane protein at work 301 10. Concluding remarks 301 11. Acknowledgements 302 12. References 302 For a variety of reasons – not the least biomedical importance – integral membrane proteins are now very much in focus in many areas of molecular biology, biochemistry, biophysics, and cell biology. Our understanding of the basic processes of membrane protein assembly, folding, and structure has grown significantly in recent times, both as a result of new methodological developments, more high-resolution structure data, and the possibility to analyze membrane proteins on a genome-wide scale. So what is new in the membrane protein field? Various aspects of membrane protein assembly and structure have been reviewed over the past few years (Cowan & Rosenbusch, 1994; Hegde & Lingappa, 1997; Lanyi, 1997; von Heijne, 1997; Bernstein, 1998); here, I will try to bring together a number of exciting recent developments. Particularly noteworthy are the discoveries related to the mechanisms of membrane protein assembly into the inner membrane of E. coli , the inner membrane of mitochondria, and the way transmembrane segments are handled by the ER translocon. Other advances include detailed studies of the interaction between transmembrane helices and the lipid bilayer, and of helix–helix packing interactions in the membrane environment. The availability of full genomic sequences have made it possible to study membrane proteins on a genome-wide scale. Finally, a handful of new high-resolution 3D structures have appeared. This review will deal only with helix bundle proteins, i.e. integral membrane proteins where the transmembrane segments form α-helices. For reviews on the other major class of integral membrane proteins – the β-barrel proteins – see Schirmer (1998) and Buchanan (1999). For readers who prefer a more ‘literary’ introduction to the membrane protein field, may I suggest von Heijne (1999).
Membrane-protein integration, folding and assembly processes in vivo depend on complex targeting, translocation, chaperoning, and sorting machineries that somehow read the ‘molecular code’ built into the nascent polypeptide, ultimately producing a properly folded protein integrated into the correct target membrane. Although the main molecular constituents and the basic mechanistic principles of many of these machines are known in outline, the codes remain poorly defined and there is little quantitative information on how protein sequence affects the final structure of membrane proteins. By carefully designing model protein constructs, we have derived the first true biological hydrophobicity scale and have been able to get a first impression of how the position of a given type of residue within a transmembrane segment affects its ability to promote membrane insertion.
We have developed reliability scores for five widely used membrane protein topology prediction methods, and have applied them both on a test set of 92 bacterial plasma membrane proteins with experimentally determined topologies and on all predicted helix bundle membrane proteins in three fully sequenced genomes: Escherichia coli, Saccharomyces cerevisiae and Caenorhabditis elegans. We show that the reliability scores work well for the TMHMM and MEMSAT methods, and that they allow the probability that the predicted topology is correct to be estimated for any protein. We further show that the available test set is biased towards high-scoring proteins when compared to the genome-wide data sets, and provide estimates for the expected prediction accuracy of TMHMM across the three genomes. Finally, we show that the performance of TMHMM is considerably better when limited experimental information (such as the in/out location of a protein's C terminus) is available, and estimate that at least ten percentage points in overall accuracy in whole-genome predictions can be gained in this way.
No abstract is provided for this article.
Trans-membrane helices have often been claimed to show “sidedness” in the distribution of polar and hydrophobic residues. However, an analysis of the statistical distribution of polar residues in randomly generated helices shows that the degree of bias commonly observed in real helices is far from statistically significant. It is concluded that “patchy” distributions of residues in such helices should be interpreted with great care.
This chapter discusses the ways in which the amino acid sequence of a membrane protein dictates its insertion into the membrane and the ways in which it determines the final three dimensional fold. The chapter also focuses on membrane protein assembly that is found in the inner membrane of the bacterium Escherichia coli (E. coli) and has led to the formulation of the “positive inside” rule. The challenge that must be addressed to understand the entire assembly process is whether the formation of the topology corresponds to a distinct step in the assembly pathway, or whether the formation of the topology and the formation of the entire 3D structure are inseparable processes. Periplasmic and outer membrane E. coli proteins normally make use of the so-called Sec machinery for translocation across the inner membrane.
Proline residues are rarely found in the three most C‐terminal positions of bacterial signal peptides, and have never been found in position +1 immediately following the cleavage site. It was recently shown that a Pro +1 mutation in the E. coli maltose binding protein precursor not only prevents cleavage of the signal peptide but also inhibits the leader peptidase enzyme, resulting in cessation of cell growth (Barkocy‐Gallagher, G.A. and Bassford, P.J. (1992) J. Biol. Chem. (in press)). Since maltose binding protein is dependent on the sec machinery for translocation across the inner membrane, it was not clear if this ‘Pro +1 ’ effect was restricted to sec ‐dependent proteins, or whether it applies also to proteins that do not require the sec functions for translocation. We now present data suggesting that the striking phenotypic effects of Pro +1 mutations can be elicited also by sec ‐independent proteins.
