Characterizing Transient Disordered Protein Structures with Time-resolved X-ray Techniques and Molecular Dynamics Simulations

Hsu, Darren Jason

doi:https://doi.org/10.21985/n2-pvsk-cv81

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Characterizing Transient Disordered Protein Structures with Time-resolved X-ray Techniques and Molecular Dynamics Simulations

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The three dimensional structure of a protein determines its biological function. How a protein folds, however, has been the central research problem for decades. The intermediates on the folding paths, which the protein can assume transiently, are of particular interest as they not only reveal the structural dynamics of a protein, but also have real-life implications; partially unfolded proteins are frequently the precursors of protein aggregates, and they in turn are linked to neurodegenerative diseases. Therefore, characterizing the transiently disordered structures is an important topic. This work documents the structural analysis of two systems, cytochrome c (cyt c) and bovine alpha-lactalbumin (BLA). In addition, modeling effort to explain the signal is detailed. For cyt c, the folding is initiated after CO is photolyzed from the heme. X-ray transient absorption (XTA) and time-resolved X-ray solution scattering (TRXSS) were utilized as experimental probes, each looking at different aspects of a protein: the active site, and the global conformational change, respectively. Combining XTA and TRXSS, a protective protein backbone during the folding that shields other ligands from replacing the native methionine was proposed, as the heme to methionine bond was found to be weak. Inspired by the study on cyt c, and in order to further explain TRXSS signal, a theoretical framework for incorporating TRXSS signal into molecular dynamics (MD) simulations was developed and tested with theoretical model systems for its efficiency and correctness. It was found that graphical processing unit (GPU) calculation significantly speeds up the calculation of TRXSS signal and the hydration shell around the protein plays a crucial role in driving the MD simulations to a correct structure. Finally, the unfolding of BLA after a temperature-jump (T-jump) was studied as a real-life application. Three intermediates were found, and their associated difference scattering patterns were incorporated into MD simulations to refine the sampled structures, which found a molten globule state matching the literature description based on optical studies and two terminal unfolded states. Overall, with the framework developed in this work, it is now possible to integrate TRXSS into the structural analysis pipeline of disordered proteins.

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