Sedimentation Velocity Analytical Ultracentrifugation

Interacting Systems

Peter Schuck, Huaying Zhao

• Publisher: CRC Press
• Year: 2017
• eBook ISBN: 9781351976831
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Biochemistry in Medicine

Analytical ultracentrifugation is one of the most powerful solution techniques for the study of macromolecular interactions, to define the number and stoichiometry of complexes formed, and to measure affinities ranging from very strong to very weak and repulsive.

Building on the data analysis tools described in the volume Sedimentation Velocity Analytical Ultracentrifugation: Discrete Species and Size-Distributions of Macromolecules and Particles, and the experimental and instrumental aspects in the first volume Basic Principles of Analytical Ultracentrifugation, the present volume Sedimentation Velocity Analytical Ultracentrifugation: Interacting Systems is devoted to the theory and practical data analysis of dynamically coupled sedimentation processes.

This volume is designed to fill a gap in biophysical methodology to provide a framework that builds on the fundamentals of the highly developed traditional methods of analytical ultracentrifugation, updated with current methodology and from a viewpoint of modern applications. It will be an invaluable resource for researchers and graduate students interested in the application of analytical ultracentrifugation in the study of interacting systems, such as biological macromolecules, multi-protein complexes, polymers, or nanoparticles.

CHAPTER 1

Exact Description of Ideally Sedimenting Associating Systems

IN this section, we focus on the description of sedimentation processes where the particles of interest exhibit interactions that can reversibly change their assembly or conformational state. This includes solutions with a single macromolecular component exhibiting self-association reactions, where homo-oligomers are formed that can dynamically dissociate back into subunits. Similarly, this includes systems with chemical binding reactions between different macromolecular components that lead to the formation of hetero-oligomers, which, in turn, can spontaneously break apart into their components. At the same time, solutions are assumed dilute enough for the limit of ‘ideal sedimentation’ to hold, such that repulsive interactions from volume exclusion and hydrodynamic interactions can still be neglected. The reduction of interactions to solely reflect either assembled or dissociated states neglects intermediate relative spatial and energetic configurations, which will be considered later in conjunction with hydrodynamic interactions and macromolecular distance distributions (Chapter 5). The utility of the ‘ideal sedimentation’ approximation is dictated by the time-scale of sedimentation, as well as the concentration range of the available detection systems.

Practical examples of systems exhibiting reversible chemical reactions in the ‘ideal sedimentation’ regime include many protein-protein and protein-nucleic acid interactions, assemblies of multi-protein complexes and molecular machines, protein-small molecule interactions, interactions involving carbohydrates, reactions between compounds of supra-molecular chemistry, and most other reactions with dissociation equilibrium constants in the micromolar range and below.

We are condensing particle characteristics to the same limited, macroscopic set of parameters as in Part II (Section 1.2) [2]. Thus, the nature of the macromolecules or particles will not play any role in the present work other than their (effective) mass, density, translational friction coefficient, and binding properties, which together determine their sedimentation behavior. For more background on how the sedimentation parameters derive, for example, the question of what can be understood as the effective sedimenting particle, considering the contributions of macromolecule-solvent and co-solute interactions, see Chapter 2 of Part I [1]. All points discussed there apply equally to interacting systems.

Sedimentation velocity offers a unique opportunity to study reversible binding reactions free in solution due to its geometric configuration where complexes can be hydrodynamically resolved, with high size-resolution generated by the universal mass-based driving force, but where the complexes are not separated during their migration from a bath of slower-sedimenting unbound species of the constituent components.¹ As a consequence, any reversibly formed complexes can be kept populated and remain in an association/dissociation reaction reflecting kinetic and equilibrium properties of the interaction. Such dynamic association/dissociation reactions can drastically change the s...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Dedication
• Table of Contents
• Foreword
• Preface
• Symbol Description
• CHAPTER 1 ▪ Exact Description of Ideally Sedimenting Associating Systems
• CHAPTER 2 ▪ Phenomenology of Sedimentation of Interacting Systems
• CHAPTER 3 ▪ Approximate Solutions for the Coupled Lamm Equations of Reacting Systems
• CHAPTER 4 ▪ Sedimentation Coefficient Distributions
• CHAPTER 5 ▪ Nonideal Sedimentation Velocity
• CHAPTER 6 ▪ Practical Analysis of Interacting Systems
• APPENDIX A ▪ Numerical Solution of Coupled Systems of Lamm Equations for Interacting Systems
• APPENDIX B ▪ Effective Particle Theory for Multi-Component Systems
• Bibliography
• Index