1. Introduction 148

2. Basics of X-ray and neutron scattering 149

2.1 Elastic scattering of electromagnetic radiation by a single electron 149

2.2 Scattering by assemblies of electrons 151

2.3 Anomalous scattering and long wavelengths 153

2.4 Neutron scattering 153

2.5 Transmission and attenuation 155

3. Small-angle scattering from solutions 156

3.1 Instrumentation 156

3.2 The experimental scattering pattern 157

3.3 Basic scattering functions 159

3.4 Global structural parameters 161

3.4.1 Monodisperse systems 161

3.4.2 Polydisperse systems and mixtures 163

3.5 Characteristic functions 164

4. Modelling 166

4.1 Spherical harmonics 166

4.2 Shannon sampling 169

4.3 Shape determination 170

4.3.1 Modelling with few parameters: molecular envelopes 171

4.3.2 Modelling with many parameters: bead models 173

4.4 Modelling domain structure and missing parts of high-resolution models 178

4.5 Computing scattering patterns from atomic models 184

4.6 Rigid-body refinement 187

5. Applications 190

5.1 Contrast variation studies of ribosomes 190

5.2 Structural changes and catalytic activity of the allosteric enzyme ATCase 191

6. Interactions between molecules in solution 203

6.1 Linearizing the problem for moderate interactions: the second virial coefficient 204

6.2 Determination of the structure factor 205

7. Time-resolved measurements 211

8. Conclusions 215

9. Acknowledgements 216

10. References 216

A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies – on ribosomes and on the allosteric protein aspartate transcarbamoylase – illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.

1. Chaperonin action – an overview 230

2. Polypeptide binding – an essential action 235

3. Recognition of non-native polypeptide – role of hydrophobicity 236

4. Crystallographic analyses of peptide binding 237

5. Topology and secondary and tertiary structure of bound substrate polypeptide – fluorescence, hydrogen exchange and NMR studies 239

6. Binding by GroEL associated with a putative unfolding action 242

7. A potential action of substrate unfolding driven by ATP/GroES binding 245

8. Folding in theciscavity 247

9. GroEL–GroES-mediated folding of larger substrate proteins by atransmechanism 249

10. Prospects for resolving the conformations and fate of polypeptide in the chaperonin reaction 251

11. References 252

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL–GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.