Allosteric regulation of protein activity is a physico-mechanical phenomenon that underlies the coordination of cellular events throughout biology. Signal transduction, metabolism, and other essential cellular processes are completely reliant on the executions of conformational and dynamic changes that enable allosteric proteins to communicate between distant sites. To understand such biological mechanisms ? and by extension to understand how to rationally alter cellular processes, either with drugs or protein engineering ? is to understand this fundamental problem of how allosteric regulation works. Yet, even though allosteric regulation has been recognized for decades and despite the recent realization that dynamics contributes to allostery, our understanding of allosteric mechanism is still at a rudimentary level. One limitation has been that the roles of dynamics in allostery have been drawn from just a few systems, most of which lack the classic indicators of functional allostery. Another limitation is that gaining accurate information on functional dynamics is experimentally challenging. To identify basic working principles of allostery, mechanisms of allosteric behavior must be observed in proteins that are ?strongly allosteric?, where allosteric movements and signatures will be more easily identified. In the long term, knowledge of allosteric mechanism will enhance protein research in general and have a huge positive impact on design of allosteric drugs and allosteric proteins. The focus of this work will be on the allosteric enzyme chorismate mutase (CM). By all considerations, this enzyme appears to be ideal for high-resolution dissective studies of its allosteric mechanisms. CM is a canonical allosteric enzyme as evidenced by a number of characteristics: it is a symmetric dimer with active sites separated by 40 ; it undergoes T-to-R conformational transitions; it exhibits homotropic allostery (Hill coefficient = 1.6); and it exhibits heterotropic allostery with small molecule effectors that modulate activity up (by Trp) or down (by Tyr). CM is 60 kDa which makes it amenable to solution NMR studies, and it is extremely soluble and durable and yields outstanding quality NMR spectra. The rich allosteric characteristics of CM will allow classical allostery to be examined experimentally using NMR and other biochemical and biophysical methods (including computations) in unprecedented detail. In this proposal, Aims 1 and 2 employ NMR, computational methods, and chemical synthesis to characterize the structural and dynamic features of apo and liganded states of CM in solution. The responses of CM to binding effectors and a transition state analog will be monitored, all towards the goal of identification of mechanisms of heterotropic long-range communication.
Aim 3 is focused on extending a novel labeling methodology for monitoring mechanisms of homotropic allostery. ?Click? chemistry will be used to covalently and specifically tether CM promoters together to stabilize samples used for studying the elusive singly ligated state. This approach will be useful for NMR studies of protein dimers in general.
Protein allostery is a fundamental phenomenon in which ligand binding at one site affects substrate activity or ligand binding at a second, distal site of the protein. Allostery is a primary mode of regulation for enzymes and other proteins that drive signal transduction and metabolism, and it is now being used in some drug development efforts. Even though countless protein structures are now known, allostery is not understood at the mechanistic level required for rational drug or protein design. The work in this project will use high- resolution NMR spectroscopy and computational methods on the classically allosteric enzyme chorismate mutase, which is particularly well-suited for NMR studies, to observe the dynamics of structure and communication pathways that compose allosteric function.
