1. Introduction 2

2. Heterotrimeric G-protein structure 3

2.1. G-protein α subunit 3

2.2. G-protein βγ dimer 8

2.3. Unique role of Gβ5 in complexes with RGS proteins 9

2.4. Heterotrimer structure 10

2.5. Lipid modifications direct membrane association 11

3. Receptor–G protein complex 11

3.1. Low affinity interactions between inactive receptors (R) and G proteins 11

3.2. Receptor activation exposes the high-affinity G-protein binding site 12

3.3. Receptor–G protein interface 14

3.4. Structural determinants of receptor–G protein specificity 15

3.5. Models of the receptor–G protein complex 17

3.6. Sequential interactions may form the receptor–G protein complex 19

4. Molecular basis for G-protein activation 19

4.1. Potential mechanisms of receptor-catalyzed GDP release 20

4.2. GTP-mediated alteration of the receptor–G protein complex 23

5. Activation of downstream effector proteins 24

5.1. Gα interactions with effectors 24

5.2. Gβγ interactions with effectors and regulatory proteins 26

6. G-protein inactivation 28

6.1. Intrinsic GTPase-activity of Gα 28

6.2. GTPase-activating proteins 30

7. Novel regulation of G-protein signaling 31

8. New approaches to study G-protein dynamics 32

8.1. Nuclear magnetic resonance spectroscopy 32

8.2. Site-directed labeling techniques 33

8.3. Mapping allosteric connectivity with computational approaches 34

8.4. Studies of G-protein function in living cells 36

9. Conclusions 37

10. References 38

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein α subunit (Gα) binds GDP and Gβγ. Receptors activate G proteins by catalyzing GTP for GDP exchange on Gα, leading to a structural change in the Gα(GTP) and Gβγ subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Gα subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Gα, how these changes may lead to subunit dissociation and allow Gα and Gβγ to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor–G protein complex and how this interaction leads to GDP release from Gα. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor–G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.

1. The ribosome 204

2. Peptide bond formation is catalyzed by RNA 205

3. Characteristics of the uncatalyzed reaction 207

4. Potential catalytic strategies of the ribosome 207

5. Experimental systems 208

6. Substrate binding in the PT center 210

7. Induced fit in the active site 211

8. pH dependence of peptide bond formation 212

9. Reaction with full-length aa-tRNA 214

10. Role of active-site residues 215

11. pH-dependent structural changes of the active site 216

12. Entropic catalysis 217

13. Role of 2′-OH of A76 in P-site tRNA 218

14. Catalysis by proton shuttling 219

15. Plasticity of the active site 220

16. Conclusions 221

17. Acknowledgments 222

18. References 222

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site – the peptidyl transferase center – is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 107-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.

1. Introduction 2

2. Sources of structural information in solid-state NMR data 5

2.1 General remarks 5

2.2 Chemical shifts, linewidths, and magic-angle spinning 6

2.3 Dipole–dipole couplings and dipolar recoupling 8

2.4 Tensor correlation techniques 12

2.5 Solid-state NMR of aligned samples 14

2.6 Indirect sources of structural information 15

2.7 Sample preparation for solid-state NMR 15

3. Levels of structure in amyloid fibrils 18

4. Molecular structure of β-amyloid fibrils 25

4.1 Self-propagating, molecular-level polymorphism in Aβ1–40 fibrils 25

4.2 Structural model for Aβ1-40 fibrils 28

4.3 Staggering of β-strands in Aβ1-40 fibrils 32

4.4 Structure of Aβ1-42 fibrils 34

4.5 Structure of fibrils formed by short β-amyloid fragments 34

4.6 Structures of non-fibrillar aggregates 35

5. Molecular structure of other amyloid fibrils 36

5.1 Ure2p10–39 and full-length Ure2p fibrils 36

5.2 TTR105–115 fibrils 38

5.3 HET-s fibrils 38

5.4 Amylin fibrils 39

5.5 PrP fibrils 39

5.6 ccβ fibrils 40

5.7 α-synuclein fibrils 40

5.8 Calcitonin fibrils 41

6. Data relevant to various proposals regarding amyloid structure 41

6.1 β-helical models for amyloid fibrils 41

6.2 Amyloid fibrils as water-filled nanotubes 42

6.3 Domain swapping in amyloid fibrils 42

6.4 The parallel superpleated β-structure model 43

6.5 α-sheet structures in amyloid fibrils 43

7. Conclusions 44

8. Acknowledgments 46

9. References 46

Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the β-amyloid peptide associated with Alzheimer's disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.

1. Introduction 326

2. Instrumentation 328

2.1 Instruments to study mechanical properties of RNA 328

2.1.1 AFM 328

2.1.2 Magnetic tweezers 328

2.1.3 Optical tweezers 330

2.2 Optical trap instrumentation 330

2.3 Calibrations 332

2.3.1 Calibration of trap stiffness 332

2.3.2 Calibration of force 333

2.3.3 Calibration of distance 334

2.4 Types of experiments 334

2.4.1 Force-ramp 334

2.4.2 Force-clamp or constant-force experiments 335

2.4.3 Extension-clamp or constant extension experiments 335

2.4.4 Force-jump, Force-drop 336

2.4.5 Passive mode 336

3. Thermodynamics 336

3.1 Reversibility 336

3.2 Gibbs free energy 337

3.2.1 Stretching free energy 338

3.2.1.1 Rigid molecules 338

3.2.1.2 Compliant or flexible molecules 339

3.2.2 Free energy of a reversible unfolding transition 339

3.2.3 Free energy of unfolding at zero force 340

3.2.4 Free energy of an irreversible unfolding transition 340

3.2.4.1 Jarzynski's method 341

3.2.4.2 Crooks fluctuation theorem 343

4. Kinetics 345

4.1 Measuring rate constants 345

4.1.1 Hopping 345

4.1.2 Force-jump, Force-drop 347

4.1.3 Force-ramp 348

4.1.4 Instrumental effects 350

4.2 Kinetic mechanisms 351

4.2.1 Free-energy landscapes 351

4.2.2 Kinetics of unfolding 353

5. Relating force-measured data to other measurements 354

5.1 Thermodynamics 354

5.2 Kinetics 357

6. Acknowledgements 357

7. References 358

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

1. Introduction 229

2. Structures 234

2.1 The structure of LH2 234

2.2 Natural variants of peripheral antenna complexes 242

2.3 RC–LH1 complexes 242

3. Spectroscopy 249

3.1 Steady-state spectroscopy 249

3.2 Factors which affect the position of the Qy absorption band of Bchla 249

4. Regulation of biosynthesis and assembly 257

4.1 Regulation 257

4.1.1 Oxygen 257

4.1.2 Light 258

4.1.2.1 AppA: blue-light-mediated regulation 259

4.1.2.2 Bacteriophytochromes 259

4.1.3 From the RC to the mature PSU 261

4.2 Assembly 261

4.2.1 LH1 262

4.2.2 LH2 263

5. Frenkel excitons 265

5.1 General 265

5.2 B800 267

5.3 B850 267

5.4 B850 delocalization 273

6. Energy-transfer pathways: experimental results 274

6.1 Theoretical background 274

6.2 ‘Follow the excitation energy’ 276

6.2.1 Bchla→Bchla energy transfer 277

6.2.1.1 B800→B800 277

6.2.1.2 B800→B850 278

6.2.1.3 B850→B850 279

6.2.1.4 B850→B875 280

6.2.1.5 B875→RC 280

6.2.2 Car[harr ]Bchla energy transfer 281

7. Single-molecule spectroscopy 284

7.1 Introduction to single-molecule spectroscopy 284

7.2 Single-molecule spectroscopy on LH2 285

7.2.1 Overview 285

7.2.2 B800 286

7.2.2.1 General 286

7.2.2.2 Intra- and intercomplex disorder of site energies 287

7.2.2.3 Electron-phonon coupling 289

7.2.2.4 B800→B800 energy transfer revisited 290

7.2.3 B850 293

8. Quantum mechanics and the purple bacteria LH system 298

9. Appendix 299

9.1 A crash course on quantum mechanics 299

9.2 Interacting dimers 305

10. Acknowledgements 306

11. References 307

This review describes the structures of the two major integral membrane pigment complexes, the RC–LH1 ‘core’ and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to ‘power’ the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

1. Prologue 58

2. The Hodgkin–Huxley formalism for computing the action potential 59

2.1 The axon action potential model 59

2.2 Cardiac action potential models 62

3. Ion-channel based formulation of the action potential 65

3.1 Ion-channel structure 65

3.2 Markov models of ion-channel kinetics 66

3.3 Role of selected ion channels in rate dependence of the cardiac action potential 71

3.4 Physiological implications of IKs subunit interaction 77

3.5 Mechanism of cardiac action potential rate-adaptation is species dependent 78

4. Simulating ion-channel mutations and their electrophysiological consequences 81

4.1 Mutations in SCN5A, the gene that encodes the cardiac sodium channel 82

4.1.1 The ΔKPQ mutation and LQT3 82

4.1.2 SCN5A mutation that underlies a dual phenotype 87

4.2 Mutations in HERG, the gene that encodes IKr: re-examination of the ‘gain of function/loss of function’ concept 94

4.3 Role of IKs as ‘repolarization reserve’ 100

5. Modeling cell signaling in electrophysiology 102

5.1 CaMKII regulation of the Ca2+ transient 102

5.2 The β-adrenergic signaling cascade 105

6. Epilogue 107

7. Acknowledgments 108

8. References 109

The cardiac cell is a complex biological system where various processes interact to generate electrical excitation (the action potential, AP) and contraction. During AP generation, membrane ion channels interact nonlinearly with dynamically changing ionic concentrations and varying transmembrane voltage, and are subject to regulatory processes. In recent years, a large body of knowledge has accumulated on the molecular structure of cardiac ion channels, their function, and their modification by genetic mutations that are associated with cardiac arrhythmias and sudden death. However, ion channels are typically studied in isolation (in expression systems or isolated membrane patches), away from the physiological environment of the cell where they interact to generate the AP. A major challenge remains the integration of ion-channel properties into the functioning, complex and highly interactive cell system, with the objective to relate molecular-level processes and their modification by disease to whole-cell function and clinical phenotype. In this article we describe how computational biology can be used to achieve such integration. We explain how mathematical (Markov) models of ion-channel kinetics are incorporated into integrated models of cardiac cells to compute the AP. We provide examples of mathematical (computer) simulations of physiological and pathological phenomena, including AP adaptation to changes in heart rate, genetic mutations in SCN5A and HERG genes that are associated with fatal cardiac arrhythmias, and effects of the CaMKII regulatory pathway and β-adrenergic cascade on the cell electrophysiological function.

1. Introduction 2

2. What is the significance of the shared structural properties of disease-associated protein fibrils? 3

2.1 Mechanism of amyloid fibril formation in vitro 6

2.1.1 In vitro fibril formation involves transient population of ordered aggregates of intermediate stability, or protofibrils 6

3. Toxic properties of protofibrils 7

3.1 Protofibrils, rather than fibrils, are likely to be pathogenic 7

3.2 The toxic protofibril may be a mixture of related species 8

3.3 Morphological similarities of protofibrils suggest a common mechanism of toxicity 9

3.4 Are the amyloid diseases a subset of a much larger class of previously unrecognized protofibril diseases? 9

3.5 Fibrils, in the form of aggresomes, may function to sequester toxic protofibrils 9

4. Amyloid pores, a common structural link among protein aggregation neurodegenerative diseases 10

4.1 Mechanistic studies of amyloid fibril formation reveal common features, including pore-like protofibrils 10

4.1.1 Amyloid-β (Aβ) (Alzheimer's disease) 10

4.1.2 α-Synuclein (PD and diffuse Lewy body disease) 12

4.1.3 ABri (familial British dementia) 13

4.1.4 Superoxide dismutase-1 (amyotrophic lateral sclerosis) 13

4.1.5 Prion protein (Creutzfeldt–Jakob disease, bovine spongiform encephalopathy, etc.) 14

4.1.6 Huntingtin (Huntington's disease) 14

4.2 Amyloidogenic proteins that are not linked to disease also from pore-like protofibrils 15

4.3 Amyloid proteins form non-fibrillar aggregates that have properties of protein channels or pores 15

4.3.1 Aβ ‘channels’ 15

4.3.2 α-Synuclein ‘pores’ 16

4.3.3 PrP ‘channels’ 16

4.3.4 Polyglutamine ‘channels’ 17

4.4 Nature uses β-strand-mediated protein oligomerization to construct pore-forming toxins 17

5. Mechanisms of protofibril induced toxicity in protein aggregation diseases 19

5.1 The amyloid pore can explain the age-association and cell-type selectivity of the neurodegenerative diseases 19

5.2 Protofibrils may promote their own accumulation by inhibiting the proteasome 20

6. Testing the amyloid pore hypothesis by attempting to disprove it 21

7. Acknowledgments 22

8. References 22

Protein fibrillization is implicated in the pathogenesis of most, if not all, age-associated neurodegenerative diseases, but the mechanism(s) by which it triggers neuronal death is unknown. Reductionist in vitro studies suggest that the amyloid protofibril may be the toxic species and that it may amplify itself by inhibiting proteasome-dependent protein degradation. Although its pathogenic target has not been identified, the properties of the protofibril suggest that neurons could be killed by unregulated membrane permeabilization, possibly by a type of protofibril referred to here as the ‘amyloid pore’. The purpose of this review is to summarize the existing supportive circumstantial evidence and to stimulate further studies designed to test the validity of this hypothesis.

1. Introduction 362

1.1 The elusive water pores 362

1.2 CHIP28 362

2. Studies on AQP-1 363

2.1 Expression of AQP1 cDNA in Xenopus oocytes 363

2.2 Reconstitution of purified AQP1 into artificial lipid bilayers 364

2.3 Structural information deduced from the primary sequence 365

2.4 Evolution and mammalian AQPs 365

3. Chronological overview over AQP structures 368

3.1 AQP1 – the red blood cell water pore 368

3.2 GlpF – the E. coli glycerol facilitator 371

3.3 AQPZ – the E. coli water pore 372

3.4 AQP0 – the lens-specific aquaporin 373

3.5 AQP4 – the main aquaporin in brain 377

3.6 SoPiP2;1 – a plant aquaporin 379

3.7 AQPM – an archaeabacterial aquaporin 379

4. Proton exclusion 380

5. Substrate selectivity 382

6. Pore regulation 385

6.1 Hormonal regulation of AQP trafficking 385

6.2 Influence of pH on AQP water conduction 386

6.3 Regulation of AQP pore conductance by protein binding 387

6.4 Pore closure by conformational changes in the AQP0 pore 388

7. Unresolved questions 390

8. Acknowledgments 390

9. References 391

The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.