Conformational Analysis of Disordered Proteins Using H/D Exchange Mass Spectrometry

Intrinsically disordered proteins (IDPs) represent a growing area of scientific interest. The prevalence of IDPs within the proteomes of complex organisms and the importance of these proteins in cellular functions has become apparent. The application of hydrogen/deuterium exchange mass spectrometry (H/D-MS) to study IDPs is presented in this dissertation. Results that demonstrate improvements to the H/D-MS method are also presented. A thermoelectrically cooled refrigeration system was developed in order to house LC components necessary for bottom-up H/D-MS. This system was used to keep solvent temperatures low and stable, ensuring reproducible and minimized back exchange. Two model IDPs, the interacting domains of CREB binding protein (CBP, residues 2059-2117) and activator of thyroid and retinoid receptors (ACTR, residues 1018-1088), were analyzed using H/D-MS. CBP and ACTR represent two classes of IDPs: the molten globule and random coil, respectively. The mutual synergistic folding phenomenon observed when a complex is formed between these two proteins was also analyzed. A lower limit of several seconds of D2O exposure time is imposed when manual pipetting is used to add label and quench buffers to protein samples. A quench flow apparatus was used to extend the lower time scale limit of exposure times to 42 milliseconds. When CBP and ACTR were labeled using the quench flow apparatus, analyses revealed subtle exchange protection in specific regions of both proteins. An H/D-MS study of the conformational dynamics of β-glucuronidase was also conducted. The destabilization of β-glucuronidase that results from the W492G mutation and the partial conformational rescue that results from indole addition were analyzed. A new method to interpret and normalize H/D-MS data was introduced to improve intra- and inter-laboratory comparability

The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja407644b.The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, x-ray crystallography and hydrogen-deuterium mass spectrometry we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure