To gain access to the largely unexplored landscapes of “folding-challenged” proteins, we are applying an approach that does not promote unfolding and aggregation: hydrogen exchange mass spectrometry (HXMS) to detect motions of native proteins as they populate their landscape at equilibrium (Jaswal & Miranker, Protein Science, 16, 2378-90, 2007). HXMS is emerging as one of the most powerful structural approaches to map the dynamics of proteins during folding, assembly into complex protein machines, transient interaction with cofactors and as substrates of chaperones and the proteasome. However, the full potential of HXMS as a universal and quantitative landscape-mapping approach has yet to be realized, as a result of practical and conceptual challenges to interpreting the data.
We are developing a HXMS approach that overcomes those limitations: combining a deconvolution algorithm that reduces the signal complexity in the spectrum, a simple analysis that extracts rough estimates of kinetic rate constants for unfolding and folding, numerical simulations that incorporate sequence and structural data to test and refine those rate constants through modeling and optimization. We are currently validating our approach by applying it to model proteins whose folding has been well characterized through traditional methods.