Transient native-state unfolding has been implicated as the initial stage of protein mis-folding and subsequent aggregation in numerous systems that propagate into a multitude of diseases. The basic goal of the proposed research is to map the native-state energy landscape of apo-myoglobin using NMR R2 dispersion techniques, with the applied goal of developing a new model system for understanding transient protein unfolding and aggregation. R2 dispersion has been successfully applied to dynamics studies of enzyme function around the active site, however, few studies have focused on the complex fluctuations accessible to a slightly perturbed native-state. 15N R2 dispersion will be applied to understand the unfolding transitions exhibited by WT apo-myoglobin at a range of pH values for which the apoMb retains its native-state structure. The identity of the excited states will be investigated, along with the pH and temperature dependence of unfolding and refolding events. Initial results suggest that under some conditions a two-site exchange occurs between the native-state, and the well characterized molten globule. Data from ~90 resonances will be fit to a local two-state mechanism, and assessed for cooperativity and mechanism. Global and cluster fitting routines will be applied to improve estimates of the exchange parameters and to identify units of cooperative structure. Multiple temperatures will be used to understand the thermodynamics of the transitions, as well as the temperature dependence of conformational exchange. Mutants will be used to probe the contribution of the dynamically fluctuating F-helix in the native-state energy landscape, while full perdeuteration of non-exchangeable protons will be used to selectively probe the contribution of the hydrophobic effect on the conformational fluctuations. WT apo-myoglobin samples will then be tuned to yield dynamics information about the processes leading to amorphous (at ~1M urea) and amyloid (at pH 9, >40? C) aggregation using 15N and 13C-methyl R2-dispersion techniques respectively. The sample conditions will be carefully controlled to maintain soluble samples while promoting the dynamics that initiate aggregation. Specific comparisons will be made to uncover differences in the mechanism of unfolding and refolding between aggregation-inducing conditions, and those which promote transient unfolding but no aggregation.
In order to understand the initial stage of many aggregation mechanisms, it is necessary to understand the process of protein unfolding. Since protein function is frequently dependent on transient unfolding, it is important to uncover the key differences between unfolding events that are not implicated in aggregation, with those which result in the formation of toxic multimeric species.
|Meinhold, Derrick W; Wright, Peter E (2011) Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion. Proc Natl Acad Sci U S A 108:9078-83|