Review Article
Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine
- JAMES C. WANG
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- Published online by Cambridge University Press:
- 01 May 1998, pp. 107-144
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The discovery of the double helix structure of DNA led immediately to questions on the mechanics of unravelling its intertwined strands during replication. If a parental DNA is to be duplicated into two progeny molecules by separating its two strands and copying each, then the strands must untwine rapidly during replication (Watson & Crick, 1953).
That DNA indeed replicates in such a semiconservative fashion was soon demonstrated by the Meselson–Stahl experiment (1958). At first, it appeared that the unravelling of the intertwined strands should not pose an insurmountable mechanical problem. The two strands at one end of a linear DNA, for example, can be pulled apart with concomitant rotation of the double-stranded portion of the molecule around its helical axis. If the strands of a DNA double helix are to separate at an estimated replication rate of 100000 base pairs (bp) per minute, then the speed of this rotation would be 10000 revolutions per minute from the 10 bp per turn helical geometry of the double helix. This speed, though impressive, seemed reasonable: owing to the slender rod-like shape of the double helix, the estimated viscous drag for this rotational motion is actually rather modest (Meselson, 1972).
A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs)
- GEOFFREY W. ABBOTT, STEVE A. N. GOLDSTEIN
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- Published online by Cambridge University Press:
- 01 November 1998, pp. 357-398
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1. INTRODUCTION 358
1.1 Summary 358
1.2 Overview 359
1.3 Four classes of pore-forming K+channel subunits – necessary and (sometimes) sufficient 361
1.4 Soluble and peripheral membrane proteins that interact with P loop subunits to alter function 362
1.5 Integral membrane proteins that interact with P loop subunits to alter function 363
2. MinK DETERMINES THE FUNCTION OF MIXED CHANNEL COMPLEXES 363
2.1 The KCNE1 gene product (MinK) gives rise to K+-selective currents and controversy 363
2.2 MinK assembles with a P loop protein, KvLQT1, to form K+channels with unique function 364
2.2.1 Single-channel conductance of KvLQT1 and MinK/KvLQT1 channels 366
2.2.2 Other differences between KvLQT1 and MinK/KvLQT1 channels 367
2.3 MinK assembles with HERG, another P loop subunit, to regulate channel activity 368
2.4 MinK does not form chloride-selective ion channels 368
3. EXPERIMENTAL AND NATURAL MinK MUTATIONS 369
3.1 Site-directed mutations 369
3.1.1 MinK mutation alters basic channel attributes and identifies key residues 369
3.1.2 MinK is a Type I transmembrane peptide 370
3.1.3 MinK is intimately associated with the IKspore 370
3.1.4 The number of MinK subunits in IKschannel complexes 372
3.2 KCNE1 mutations associated with arrhythmia and deafness alter IKschannel function 373
3.3 Summary of MinK sites critical to IKschannel function 374
4. MinK-RELATED PEPTIDES: AN EMERGING SUPERFAMILY 374
4.1 KCNE2, 3 and 4 encode MinK-related peptides 1, 2 and 3 (MiRPs) 374
4.2 MiRP1 assembles with a P loop protein, HERG, to form K+channels with unique function 375
4.2.1 MiRP1 alters activation, deactivation and single-channel conductance 376
4.2.2 MiRP1 alters regulation by K+ion and confers biphasic kinetics to channel blockade 378
4.2.3 Stable association of MiRP1 and HERG subunits 380
4.3 KCNE2 mutations are associated with arrhythmia and decreased K+flux 383
4.4 Summary of the evidence that cardiac IKrchannels are MiRP1/HERG complexes 385
5. MinK-RELATED PEPTIDES: COMMONALTIES AND IMPLICATIONS 386
5.1 Genetics and structure 386
5.2 Cell biology and function 387
6. ANSWERS, SOME OUTSTANDING ISSUES, CONCLUSIONS 387
7. ACKNOWLEDGEMENTS 389
8. REFERENCES 389
MinK and MinK-related peptide 1 (MiRP1) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K+ channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRP1/HERG complexes reproduce the cardiac currents called IKs and IKr, respectively. Inherited mutations in KCNE1 (encoding MinK) and KCNE2 (encoding MiRP1) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily.
The moving parts of voltage-gated ion channels
- GARY YELLEN
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- Published online by Cambridge University Press:
- 01 August 1998, pp. 239-295
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Ion channels, like many other proteins, have moving parts that perform useful functions. The channel proteins contain an aqueous, ion-selective pore that crosses the plasma membrane, and they use a number of distinct ‘gating’ mechanisms to open and close this pore in response to biological stimuli such as the binding of a ligand or a change in the transmembrane voltage.
This review is written at a watershed in our understanding of ion channels.
1. INTRODUCTION 240
1.1 Basic structure of voltage-activated channels 241
1.2 What are the physical motions of the channel protein during gating? 243
1.3 Gating involves several distinct mechanisms of activation and inactivation 246
2. ACTIVATION GATING 246
2.1 Early evidence for an activation gate at the intracellular mouth 247
2.1.1 Open channel blockade 247
2.1.2 The ‘ foot-in-the-door’ effect 249
2.1.3 Trapping of blockers behind closed activation gates 249
2.2 Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects 250
2.3 State-dependent cysteine modification as a reporter of position and motion 251
2.4 Localization of activation gating 254
2.4.1 The trapping cavity 254
2.4.2 The activation gate 255
2.4.3 Is there more than one site of activation gating? 258
3. INACTIVATION GATING 259
3.1 Ball-and-chain (N-type) inactivation 261
3.1.1 Nature of the ‘ball’ – a tethered blocking particle 262
3.1.2 The ball receptor 263
3.1.3 The chain 264
3.1.4 Variations on the N-type inactivation theme: multiple balls, foreign balls, anti-balls 265
3.2 C-type inactivation 266
3.2.1 C-type inactivation and the outer mouth of the K+channel 266
3.2.2 The selectivity filter participates in C-type inactivation 267
3.2.3 A consistent structural picture of C-type inactivation 269
3.3 By what mechanism do other voltage-gated channels inactivate? 272
4. THE VOLTAGE SENSOR 273
4.1 Quantitative principles of voltage-dependent gating 276
4.2 S4 (and its neighbours) as the principal voltage sensor 277
4.2.1 Mutational effects on voltage-dependence and charge movement 277
4.2.2 Evidence for the translocation of S4 279
4.2.3 Real-time monitoring of S4motion by fluorescence 282
4.3 Coupling between the voltage sensor and gating 283
5. CONCLUSION 284
6. ACKNOWLEDGEMENTS 287
7. REFERENCES 287
From the globular to the fibrous state: protein structure and structural conversion in amyloid formation
- MARGARET SUNDE, COLIN C. F. BLAKE
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- Published online by Cambridge University Press:
- 01 February 1998, pp. 1-39
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The term ‘amyloid’ was used originally to describe certain deposits found post- mortem in organs and tissues, which gave a positive reaction when stained with iodine (Virchow, 1854). Only later was it realized that the material was in fact predominantly proteinaceous, although it is known to be associated with carbohydrates, particularly glucosoaminoglycans, when obtained from many ex vivo sources. With the increasing precision in the definition of amyloid, initially from its characteristic green birefringence when stained with the dye Congo Red (Missmahl & Hartwig, 1953), and later from its particular appearance under the electron microscope (Cohen & Calkins, 1959) and its X-ray diffraction pattern (Eanes & Glenner, 1968), it has become evident that it is a specific fibrillar protein state, which can also be formed by some proteins when denatured in vitro (Burke & Rougvie, 1972), and by synthetic oligopeptides (Bradbury et al. 1960) that may form amyloid spontaneously when placed in pure aqueous medium (Serpell, 1996). Although these latter may form useful experimental systems for the study of amyloid, its major interest at present is that it is associated with a number of prominent lethal diseases (Benson & Wallace, 1989; Pepys, 1994).
13C-NMR, MS and metabolic flux balancing in biotechnology research
- THOMAS SZYPERSKI
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- Published online by Cambridge University Press:
- 01 February 1998, pp. 41-106
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The European Federation of Biotechnology defines biotechnology as ‘the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services’. Biotechnology thus focuses on the industrial exploitation of biological systems and is based on their unique expertise in specific molecular recognition and catalysis. The enormous potential for drug synthesis, design of biomedical diagnostics, large-scale production of biochemicals including fuels, food production, degradation of resistant wastes and extraction of raw materials will very likely make biotechnology, along with electronics and material sciences, one of the key technologies of the 21st century. From the chemical engineer's point of view, the living system participating in a biotechnological process is the central unit that catalyses chemical reactions. It exhibits a complex dependence on the bioprocess parameters, and the engineer focuses on these parameters to achieve optimal control (Hamer, 1985; Bailey & Ollis, 1986). For the natural scientist, the living system itself is in the centre of interest, so that attempts to optimize a bioprocess aim at its appropriate redesign by genetic manipulations. The increase in penicillin production by strain improvement based on random mutagenesis, which was pursued from 1940 to the mid 1970s, represents an early contribution of life scientists to improve a bioprocess that is of utmost medical importance (Hardy & Oliver, 1985).
Structure calculation of biological macromolecules from NMR data
- PETER GÜNTERT
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- Published online by Cambridge University Press:
- 01 May 1998, pp. 145-237
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The relationship between amino acid sequence, three-dimensional structure and biological function of proteins is one of the most intensely pursued areas of molecular biology and biochemistry. In this context, the three-dimensional structure has a pivotal role, its knowledge being essential to understand the physical, chemical and biological properties of a protein (Branden & Tooze, 1991; Creighton, 1993). Until 1984 structural information at atomic resolution could only be determined by X-ray diffraction techniques with protein single crystals (Drenth, 1994). The introduction of nuclear magnetic resonance (NMR) spectroscopy (Abragam, 1961) as a technique for protein structure determination (Wüthrich, 1986) has made it possible to obtain structures with comparable accuracy also in a solution environment that is much closer to the natural situation in a living being than the single crystals required for protein crystallography.
Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins
- MATTHIAS BUCK
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- Published online by Cambridge University Press:
- 01 August 1998, pp. 297-355
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Alcohol based cosolvents, such as trifluoroethanol (TFE) have been used for many decades to denature proteins and to stabilize structures in peptides. Nuclear magnetic resonance spectroscopy and site directed mutagenesis have recently made it possible to characterize the effects of TFE and of other alcohols on polypeptide structure and dynamics at high resolution. This review examines such studies, particularly of hen lysozyme and β-lactoglobulin. It presents an overview of what has been learnt about conformational preferences of the polypeptide chain, the interactions that stabilize structures and the nature of the denatured states. The effect of TFE on transition states and on the pathways of protein folding and unfolding are also reviewed. Despite considerable progress there is as yet no single mechanism that accounts for all of the effects TFE and related cosolvents have on polypeptide conformation. However, a number of critical questions are beginning to be answered. Studies with alcohols such as TFE, and ‘cosolvent engineering’ in general, have become valuable tools for probing biomolecular structure, function and dynamics.
1. COSOLVENTS: OLD HAT? 298
2. HOW DOES TFE WORK? 299
2.1 Effect on hydrogen bonding 300
2.2 Effect on non-polar sidechains 301
2.3 Effect on solvent structure 302
3. EFFECTS OF TFE ON (UN-)FOLDING TRANSITIONS 303
3.1 Pretransition 303
3.2 Transition 304
3.3 Posttransition 305
3.4 Far UV CD spectroscopic detection of structure 306
3.5 Effect with temperature 306
3.6 Effect with additional denaturants 306
4. THERMODYNAMIC PARAMETERS FROM STRUCTURAL TRANSITIONS OF PEPTIDES AND PROTEINS IN TFE 307
5. ADVANCES IN NMR SPECTROSCOPY 310
5.1 Chemical shifts 310
5.2 3[Jscr ]HNHαcoupling constants 311
5.3 Amide hydrogen exchange 312
5.4 Nuclear Overhauser Effects (NOEs) 312
6. α-HELIX – EVERYWHERE? 313
6.1 Intrinsic helix propensity equals helix content? 313
6.2 A helix propensity scale for the amino acids in TFE 314
6.3 Capping motifs and stop signals 315
6.4 Limits and population of helices as seen by CD and NMR 316
7. TURNS 317
8. β-HAIRPINS AND SHEETS 317
9. ‘CLUSTERS’ OF SIDECHAINS 320
10. THE TFE DENATURED STATE OF β-LACTOGLOBULIN 321
11. THE TFE DENATURED STATE OF HEN LYSOZYME 324
12. TERTIARY STRUCTURE, DISULPHIDES, DYNAMICS AND COMPACTNESS 327
13. PROSPECTS FOR STRUCTURE CALCULATION 328
14. EFFECT OF TFE ON QUATERNARY STRUCTURE 329
15. EFFECT ON TFE ON UN- AND REFOLDING KINETICS 330
16. OTHER USES 336
16.1 Mimicking membranes and protein receptors 336
16.2 Solubilizing peptides and proteins 336
16.3 Cosolvents as helpers for protein folding? 338
16.4 Modifying protein dynamics and catalysis 338
16.5 Effects on nucleic acids 339
16.6 Effects on lipid bilayers and micelles 339
16.7 Future applications 339
17. CONCLUSIONS: TFE – WHAT IS IT GOOD FOR? 340
18. ACKNOWLEDGMENTS 340
19. REFERENCES 340
Conserved geometrical base-pairing patterns in RNA
- NEOCLES B. LEONTIS, ERIC WESTHOF
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- Published online by Cambridge University Press:
- 01 November 1998, pp. 399-455
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1. INTRODUCTION 399
2. DEFINITIONS 401
3. CIS BASEPAIRS 410
3.1 Cis Watson–Crick/Watson–Crick 410
3.2 Wobble pairings 411
3.3 Cis Watson–Crick/Hoogsteen pairings 416
3.4 Bifurcated pairings 417
3.5 Cis open and water-inserted 421
4. TRANS BASEPAIRS 423
4.1 Trans Watson–Crick/Watson–Crick 423
4.2 Trans wobble pairs 424
4.3 Trans Watson–Crick/Hoogsteen pairs 424
4.4 Trans Hoogsteen/Hoogsteen pairs 430
4.5 Trans bifurcated pairings 432
5. SHALLOW-GROOVE PAIRINGS 432
5.1 Hoogsteen/Shallow-groove pairs 433
5.2 Watson–Crick/Shallow-groove pairings 438
5.3 Shallow-groove/Shallow-groove pairings 440
6. SIDE-BY-SIDE BASES 446
7. DEFINING A LIBRARY OF ISOSTERIC PAIRINGS 446
8. CONCLUSIONS 451
9. ACKNOWLEDGEMENTS 452
10. REFERENCES 452
RNA molecules fold into a bewildering variety of complex 3D structures. Almost every new RNA structure obtained at high resolution reveals new, unanticipated structural motifs, which we are rarely able to predict at the current stage of our theoretical understanding. Even at the most basic level of specific RNA interactions – base-to-base pairing – new interactions continue to be uncovered as new structures appear. Compilations of possible non-canonical base-pairing geometries have been presented in previous reviews and monographs (Saenger, 1984; Tinoco, 1993). In these compilations, the guiding principle applied was the optimization of hydrogen-bonding. All possible pairs with two standard H-bonds were presented and these were organized according to symmetry or base type. However, many of the features of RNA base-pairing interactions that have been revealed by high-resolution crystallographic analysis could not have been anticipated and, therefore were not incorporated into these compilations. These will be described and classified in the present review. A recently presented approach for inferring basepair geometry from patterns of sequence variation (Gautheret & Gutell, 1997) relied on the 1984 compilation of basepairs (Saenger, 1984), and was extended to include all possible single H-bond combinations not subject to steric clashes. Another recent review may be consulted for a discussion of the NMR spectroscopy and thermodynamic effects of non-canonical (‘mismatched’) RNA basepairs on duplex stability (Limmer, 1997).