Accelerated Molecular Dynamics Simulations to Support Rosetta-based Protein Design
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Computational protein design offers exciting new opportunities to test our knowledge of protein folding and the thermodynamics of protein folding. Moreover, molecular dynamics (MD) simulations promise the ability to computationally probe the stability of newly designed proteins and can thus be considered as an in silico screening tool. This may allow for the selection of stable designs before a costly attempt is made to express and biophysically characterize a new design. The primary focus of the research presented in this thesis was to implement, explore and compare MD protocols using the GROMACS program package in order to determine if proteins designed by the ROSETTA program package are stable. Specifically, protocols to simulate thermal unfolding by applying a temperature gradient during the simulation, as well as, protocols to employ the ‘adaptive flooding’ approach were established, along with protocols for efficient analysis of MD trajectories. Applications are presented for proteins which were engineered in a collaborative effort by the Szyperski and Kuhlman laboratories in order to test key hypothesis related to the thermodynamics of protein folding such as the correlation of the surface area of hydrophobic and hydrophilic core residues which become water exposed upon unfolding on unfolding and the difference of the heat capacities of unfolded and folded states. The results of the MD simulations were compared with insights obtained from biophysical characterization using Nuclear Magnetic Spectroscopy (NMR) and Circular Dichroism (CD). The MD simulations turned out to be a valuable tool to assess the stability of hydrogen bond networks designed within the core of 4-helix bundles, and the adaptive flooding approach appears to be promising to assess the foldedness of 4-helix bundles with ‘underpacked’, that is, destabilized cores. The protocols presented in this thesis are now available for routine use by protein designer.