A Brief Introduction to the Analytical Ultracentrifugation of Proteins for Beginners

DAVID J. SCOTT AND PETER SCHUCK

1 Introduction

Analytical ultracentrifugation (AUC) is one of the classical methods for the characterization of purified proteins in dilute solutions, and as such there is a large body of theoretical and practical studies in the literature going back eight decades, covering aspects including the technical implementation of AUC, the theoretical foundation of ultracentrifugation in thermodynamics and physical chemistry of macromolecules, and the mathematical analysis of ultracentrifugation experiments. Developed in the 1920s by The Svedberg, and redesigned into a commercial instrument by Edward Pickels, it became a central technique in the development of biochemistry and molecular biology. While the technique was in decline in the 1970s and 1980s, new instrumentation and numerical analysis in the 1990s stimulated renewed interest in AUC, in particular for the study of protein interactions.

For the novice, however, the highly developed technical aspects in conjunction with the multitude of analytical approaches can be daunting. Therefore, this introduction to the book is unashamedly aimed at the novice who has come into contact with the methodology of the analytical ultracentrifuge and wishes to use the instrument for the characterization of globular protein samples in solution. Such a novice can be a molecular biologist, a protein crystallographer or NMR spectroscopist, or working as part of a high-through-put proteomics project, with interest in the characterization of the aggregation state, heterogeneity and thermodynamic characterization of reversible interactions of proteins. The aim of the present introduction is to provide help in where to start, or how to design and analyse a successful AUC experiment. Necessarily, this requires a very selective presentation, and for more in-depth information and descriptions of selected sedimentation velocity (SV) and sedimentation equilibrium (SE) methods, the reader is referred to recent reviews, monographs, practical protocols, and websites, and, of course, subsequent chapters in this book. Fortunately, with regard to the data interpretation, the mathematical details of advanced analysis methods are largely encapsulated in software. However, in addition to general knowledge in modelling of data and non-linear regression, their use requires understanding of the basic concepts behind them.

2 What Can I Do with My Protein Sample?

At its most basic level, AUC simply consists of the application of a centrifugal force with the simultaneous real-time observation of the resulting redistribution of the macromolecule. Analysis can be performed from first principles, given quantitative and rigorous data on a particular sample. This requires no label or other chemical modification of the proteins; such as would occur in cross-linking or many fluorescence experiments; and no interaction with any matrix or surface, as would be required for gel filtration or surface plasmon resonance. As proteins are studied in solution, the experimentalist has direct access to their solution properties: a key strength of the technique. A central feature of sedimentation experiments for the study of protein interactions is that faster sedimenting complexes are transported through a solution of the slower sedimenting components. Consequently, reversibly formed complexes that dissociate can readily re-associate during the experiment, thus permitting the hydrodynamic and thermodynamic characterization of even weak and transient interactions.

Analytical ultracentrifugation of non-interacting proteins can reveal the molar mass, gross shape, and the heterogeneity of the sample. The latter includes the detection of even trace quantities of oligomers and aggregates, which can be of interest in biotechnology applications or aid in the interpretation of biosensor experiments. For interacting systems, protein complexes can be characterized with regard to their stoichiometry and the thermodynamic and kinetic constants of complex formation....