Finding the right balance – a personal journey from individual proteins to membrane‐embedded motors

Abstract

It is now more than 20 years since the prophetic words of John Fenn, announcing his discovery, stated that ‘Electrospray spectra have been obtained for biopolymers including oligonucleotides and proteins, the latter having molecular weights up to 130 000, with as yet no evidence of an upper limit’ (Fenn JB, Mann M, Kai Meng C, Fu Wong S & Whitehouse CM (1989) Science 246, 64–71). Today, with the mass spectra of intact ribosomes at 2.3 MDa becoming almost routine and the first electrospray spectra of membrane‐embedded motors being recorded recently, new challenges are emerging. Knowledge of the intact mass of a protein or complex is only part of the MS information available. Data from the disruption of protein complexes in solution and gas phases are leading to subunit interaction maps and architectural models. Such models are enhanced by coupling with ion mobility in which the collision cross‐section of a protein complex can be defined. Linking these attributes with knowledge of subunit dynamics and the role of post‐translational modifications on the stability and interactions within complexes is increasing our understanding of the factors that stabilize and convert protein complexes between different quaternary states. From our earliest experiments, studying the folding of individual proteins, through to the characterization of membrane‐embedded motors, it is clear that the full potential of electrospray in structural biology has yet to be realized. The present review offers a personal view of the transition from determining the mass of an individual protein to elucidating the structure and dynamics of heterogeneous assemblies in the megadalton mass range.