Developing ex vivo structural biology using natural abundance NMR: the role of conformational dynamics in regulating protein metastability

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The efficient folding of proteins into their correct three-dimensional structures is essential for cellular function. In most cases this corresponds to the energetically most favourable state, but a number of metastable proteins fold instead to high energy conformations, which are primed to undergo large scale structural transformations when later required according to the particular function of the protein. Serpins are one such class of metastable proteins, of which the plasma glycoprotein alpha1-antitrypsin (AAT) is the prototypical example. Serpins comprise the most abundant family of protease inhibitors, and possess a molecular structure that is inherently dynamic: in the process of inhibiting their substrate protease, they undergo a dramatic change in shape from their initial metastable conformation. Clearly then, metastability is central to serpin function and conformational changes must be able to be triggered efficiently when required, and yet it is also their Achilles heel: spontaneous transitions can lead to misfolding or formation of polymeric aggregates, a process that is often associated with disease. Of the 35 serpin genes found in humans, nearly a third have a known involvement in hereditary disease, and five are known to form protein aggregates called polymers. However, despite many years of research the molecular mechanisms by which these transformations can be regulated remain poorly understood. It is our hypothesis that small-scale fluctuations ('dynamics') in the structure of the metastable native state may hold the key to this puzzle, and so this project is designed first to characterise the solution-state structure and dynamics of AAT molecules, and then to correlate these observations with the measured rates of conformational change.Nuclear magnetic resonance (NMR) spectroscopy is an exceptionally powerful experimental technique for studying the structure and dynamics of proteins. However, NMR traditionally requires proteins to be expressed recombinantly within bacterial cells using specialised isotopic labelling techniques, and for a number of interesting molecules, including several variants of AAT, this is not currently possible. Instead, our preliminary data overturn this paradigm by showing we can measure high quality NMR spectra using AAT purified directly from human donors - including patients with rare, disease-associated mutations - without the need for isotopic labelling. Thus, for the first time we can study the solution-state structure and dynamics of ex vivo, natively glycosylated AAT molecules, and this has revealed widespread changes in the conformation of a disease-associated variant that were not observed using crystallographic approaches that confine molecules into a rigid lattice structure. We propose to pursue these observations further, developing a new toolkit of NMR experiments to characterise structure and dynamics in these unlabelled ex vivo protein samples. We will investigate in detail the impact that mutations - associated with disease, or artificially designed - can have upon the structure and dynamics of the metastable serpin fold, and compare this with the effect the mutations have on both inhibitory activity and the misfolding and polymerisation processes.In correlating the solution structure and dynamics of AAT variants with serpin function and dysfunction, our research will address the longstanding problem of how structural changes within metastable proteins can be regulated, and this may ultimately lead to a new mechanistic basis for the design of inhibitors of serpin misfolding. More broadly, the new NMR methodologies that we will develop in this project will provide a platform that can be readily extended to ex vivo structural biology of other previously inaccessible protein systems.