24] Probing conformations of GroEL-bound substrate proteins by mass spectrometry

Abstract

Publisher Summary The chapter describes the methodology developed to elucidate electrospray ionization mass spectrometry (ESI-MS) and the conformational properties of substrate proteins bound to GroEL. To provide an overview of the information obtained with this methodology, selected examples are used. The chapter also discusses the general application of ESI-MS in the study of noncovalent protein complexes and the future potential of this rapidly advancing technique. The essential features of the conventional electrospray process are summarized. The essential procedures used to prepare complexes of GroEL with [3-SS] BLA or dihydrofolate reductase (DHFR) for analysis by ESI-MS are summarized. One important attribute of ESI-MS from native conditions is the ability to preserve noncovalent protein-ligand interactions within the mass spectrometer, and many examples now exist of protein-ligand interactions that have survived in the gas phase. The chapter discusses mass spectrometric methods for studying native and partially folded states of proteins, measurements of hydrogen exchange by electrospray ionization mass spectrometry, preparing protein samples for hydrogen exchange electrospray ionization mass spectrometry and interaction of GroEL with nucleotides.