Infrared experiments using the SCN probe group at six sites in the regulatory protein calmodulin reveal what is potentially new information about the changing conformational distribution as the protein binds calcium and then its protein targets, but quantitative interpretation of the probe IR signals has been difficult without a direct connection to MD simulations. Published results provide that direct connection for substrate-bound calmodulin, where the conformational distribution is very narrow and rigid.  Putative but experimentally unvalidated conformational distributions for both holo- calmodulin and apo-calmodulin were also published recently, and our goal is to use the IR results to validate and re-weight the simulated ensembles, starting with holo-calmodulin (where the proteins binds four calcium ions).<br/>Our general strategy is to run short MD simulations on each of six assigned structural clusters, for each probe site with correct weighting of the most likely probe dihedral orientations.  The “SolEFP” fragment potential-based approach (developed by the Cho group in South Korea) is used to calculate the frequency shift due to different physical pieces of the environment around the probe for each frame of the MD simulations. The data from SolEFP calculations in each cluster are used to create spectral lineshapes for each probe site.<br/>Comparison of the simulated and experimental lineshapes directly ties simulation to previous experiments and suggests which structural clusters contribute the most to the overall structural ensemble. A similar approach has been adopted for more dynamic complexes of viral disordered proteins with their natural binding partners; comparison between IR experimental data and simulated lineshapes can help to reweight and validate the complex's ensemble of structures. The overall goal of this methodology is to offer more information on how vibrational spectroscopy can validate the conformational distributions of various types of proteins, especially dynamic ones where other techniques can’t directly reveal the entire ensemble of structures.

MD simulations and vibrational probe frequency calculations to refine the protein structural ensembles

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.