Mass spectrometry at the frontiers of molecular medicine

Many of our vital cellular processes are controlled and carried out by proteins that come together to form large and dynamic machineries. As cornerstones of the proper functioning of the cell, unsurprisingly many of these protein assemblies play important roles in human health. These range from ensuring that other proteins are correctly folded and assembled; to being responsible for clearing and degrading toxic species; and allowing the correct movement of molecules and ions in and around the cell. As such many of these protein machineries are important drug targets, with the binding of these small molecules acting to stimulate or reduce their activity, and hence mitigate disease states. Obtaining a complete inventory and description of these machineries, in both healthy and disease states, therefore represents one of the continuing grand challenges of biology. The difficulty in studying the structure of these molecules is that they are often very dynamic, that is their composition and shape can change, particularly when they interact with other molecules or are subject to external stresses. Infact the nature, scale and frequency of these fluctuations themselves play a vital part in governing function. Here we propose to develop and apply mass spectrometry for the study of both the structure and dynamics of protein assemblies, and will tackle two of the most notoriously difficult classes of biomolecules: the molecular chaperones, involved in protecting proteins from unwanted interactions; and membrane complexes that regulate the transport of small molecules into and out of the cell. Mass spectrometry represents a particularly attractive approach as it is remarkably general in its applicability, and has a high resolution of separation in both space and time: in other words it is well suited to looking at mixtures of proteins, and to monitor the transitions they undergo. The output of this research will lead us towards a complete understanding of the sizes, shapes and movements of these cellular machineries, as well as a means for evaluating the effects of small molecules as potential therapeutic strategies.

Much progress has been made in obtaining atomic images of the important machineries that govern protein folding, degradation and transport across the cell membrane. Complementary information is required however to probe the dynamic changes in organization and conformation that these protein assemblies undergo. We propose to develop and apply mass spectrometry based approaches to a range of biomolecules that are refractory to conventional structural approaches. We plan to develop our methodology to overcome two of the most challenging characteristics displayed by protein complexes: intrinsic heterogeneity; and association with membranes. The difficulties these pose is exemplified in two systems: the polydisperse small heat shock proteins chaperones that play a crucial role in maintaining protein homeostasis; and the membrane channels and transporters that play an active role in small molecule/drug transport. The small heat shock proteins are implicated in a range of protein deposition diseases, with specific variants directly linked to cataract, desmin-related cardiomyopathy and Charcot-Marie-Tooth disease. The membrane channels and transporters we will study have important implications in type II diabetes, and the oral bio-availability of drugs. Through the basic biomedical research programme outlined here we will probe the molecular properties of these assemblies with a particular focus on monitoring their oligomeric state, heterogeneity, dynamics and conformational changes; and the ways in which these are altered in response to small molecule binding. We plan to implement the results of these fundamental studies to gain insight into the mechanism of ligand binding and its effects on these dynamic properties. We anticipate that the outcomes of this research will be a new means of studying these intractable biomolecules that will be broadly applicable to studying the effects of small molecules in polydisperse and membrane associated assemblies with high impact for biomedical research.