1. Introduction 286

2. Membrane protein assembly inE. coli286

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).

1. Introduction 310

2. Protein-only hypothesis 312

3. The scrapie prion protein PrPSc313

3.1 Purification of PrP 27–30 313

3.2 Proteinase K resistance 314

3.3 Scrapie-associated fibrils 314

3.4 Smallest infectious unit 316

3.5 Conformational properties 316

3.6 Dissociation and stability 319

4. The cellular prion protein PrPC321

4.1 Prnp expression 321

4.2 Biosynthetic pathway 322

4.3 NMR structures 324

4.4 Copper binding 326

5. Post-translational PrP conversion 327

5.1 Conformational isoforms 327

5.2 Location of propagation 329

5.3 Minimal PrP sequence 330

5.4 Prion species barrier 331

5.5 Prion strains 332

6. Effect of familial TSE mutations 333

6.1 Thermodynamic stability of PrPC 334

6.2 De novo synthesis of PrPSc 335

6.3 Transmembrane PrP forms 337

7. Physical properties of synthetic PrP 337

7.1 Amyloidogenic peptides 337

7.2 Folding intermediates 339

8. Hypothetical protein X 340

8.1 Two species-specific epitopes 340

8.2 Mapping the protein X epitope 341

9. Chaperone-mediated PrP conversion 343

9.1 Hsp60 and Hsp10 chaperonins 343

9.2 GroEL promoted PrP-res formation 345

9.3 Membrane-associated chaperonins 345

9.4 Preference of GroEL for positive charges 347

9.5 Potential GroEL/Hsp60 epitopes on PrP 347

9.6 Conformations of chaperonin-bound PrP 349

9.7 Conserved Hsp60 substrate binding sites 349

9.8 Requirement of ATP-hydrolysis 351

9.9 Hsp60-mediated prion propagation 354

10. Template-assisted annealing model 355

11. Acknowledgments 357

12. References 357

Although the central paradigm of protein folding (Anfinsen, 1973), that the unique three-dimensional structure of a protein is encoded in its amino acid sequence, is well established, its generality has been questioned due to two recent developments in molecular biology, the ‘prion’ and ‘molecular chaperone’. Biochemical characterization of infectious scrapie material causing central nervous system (CNS) degeneration indicates that the necessary component for disease propagation is proteinaceous (Prusiner, 1982), as first outlined by Griffith (1967) in general terms, and involves a conversion from a cellular prion protein, denoted PrPC, into a toxic scrapie form, PrPSc, which is facilitated by PrPSc acting as a template for PrPC to form new PrPSc molecules (Prusiner, 1987). The ‘protein-only’ hypothesis implies that the same polypeptide sequence, in the absence of any post-translational modifications, can adopt two considerably different stable protein conformations (Fig. 1). Thus, in the case of prions it is possible, although not proven, that they violate the central paradigm of protein folding. There is some indirect evidence that another factor, provisionally named ‘protein X’, might be involved in the conformational conversion process (Prusiner et al. 1998), which includes a dramatic change from α-helical into β-sheet secondary structure (Fig. 1). This factor has not been identified yet, but it has been proposed that protein X may act as a molecular chaperone. The idea that molecular chaperones play a critical role in the generation of PrPSc is appealing also from a theoretical point of view, because PrPSc formation involves changes in protein folding and possibly intermolecular aggregation (Fig. 1), processes in which chaperones are known to participate (Musgrove & Ellis, 1986). The discovery and functional analysis of more than a dozen molecular chaperones made it clear that these proteins do not complement folding information that is not already contained in the genetic code (Ellis et al. 1989); rather they facilitate the folding and assembly of proteins by preventing misfolding and refolding misfolded proteins (Hartl, 1996). Whether a molecular chaperone or another type of macromolecule is identified as the conversion factor, therefore, the molecular chaperone concept is likely to contribute to the understanding of the molecular nature of PrPC to PrPSc conversion.

In this review I consider the prion concept from the view of a structural biologist whose main interest focuses on spontaneous and chaperone-mediated conformational changes in proteins.