23] Molecular chaperones and their interactions investigated by analytical ultracentrifugation and other methodologies

Abstract

Publisher Summary The chapter presents a study related to molecular chaperones and their interactions through analytical ultracentrifugation and other methodologies. After a brief introduction into basic principles of the technique, the chapter presents practical examples to illustrate the ability of the methodology to resolve controversial questions in the chaperone field and discusses some results in the context of other methodologies used to determine quaternary structure. In an analytical ultracentrifugation experiment an analysis is made of the distribution of molecules according to their masses within a field of gravitation. This analysis can also be done using a preparative centrifuge, where the concentration gradient is measured after the run and drop fractionation of the contents of the tubes; however, in this case only final concentration profiles are obtained from a single centrifugation run and sedimentation must occur in the presence of preformed density gradients (for example, of glycerol or sucrose). The chapter discusses that determination of protein quaternary structure should never be based exclusively on relative methods. Only a few methodologies, such as analytical ultracentrifugation techniques, unambiguously deliver molecular masses of homo- or hetero-complexes in solution without the need for arbitrary assumptions. The chapter demonstrates the practicality of investigating the comparably large chaperonin heterocomplexes under equilibrium. The chapter concludes by stating that the investigation of chaperonin-substrate interactions will be a productive field for future analytical ultracentrifugation experiments.