Conformational Fluctuations of Biomolecules Studied Using Molecular Dynamics and Enhanced Samplin

Conformational fluctuations of biomolecules regulate their biological function. Changes in conformational fluctuations result in changes in the structural ensemble for a biomolecule. This in turn changes the microscopic behavior of the system and the macroscopic observables. My research has shed light on several interesting cases. Simulation of the HPV type 6 E2 protein (chapter 2) revealed the importance of the beta2-beta3 loop fluctuations that act to drive the higher affinity of the DeltaLL mutant for noncognate DNA sequences. Via rearrangements, this loop forms contacts with the DNA, which had previously not been identified experimentally. Formation of these novel contacts helped drive the increase in the binding affinity of the mutant protein. It was further discovered that the removal of the terminal Leu perturbed the beta-barrel fluctuations, allowing for a quasiharmonic mode motion in the mutant systems and the WT system bound to the cognate sequence, which was not present in the WT bound to the noncognate sequence. This in turn helped facilitate the rearrangement of the loop. Additionally, my simulations validated the previous hypothesis that the cognate DNA sequences exist in a prebent conformation compared to nongcognate sequence. This results in a lower deformation energy for the binding of a cognate sequence. Simulations of the C-terminal domain of Cdc37 (chapter 3) in which Y298 was unphosphorylated, phosphorylated and mutated to a phosphomimetic residue, revealed the importance of conformational fluctuations in the regulation of the Hsp90 chaperone cycle. Both the phosphomimetic and phosphorylated systems resulted in the loss of native like contacts and hydrogen bonding for that residue. This resulted in an unfolding of 3 1 helix and an exposure of an SH2-binding domain. This domain is important for the recognition of client chaperones. Contrastingly, while 31 helixdid unfold for the WT system, native-contacts were maintained throughout the simulation and the exposure (as measured by the solvent-accessible surface area) of the SH2-domain remained lower than the other two systems. This reveals the structural effects of Y298 phosphorylation on the Cdc37 domain and the regulatory effects of this post-translational modification on the Hsp90 chaperone cycle. The effects of the conformational fluctuations of cyclization were investigated on a novel gamma-AApeptide library (chapter 4). Previous results have indicated that there are fewer low energy conformers for cyclical peptoids resulting in a reduction in conformer space. This in turn results in a higher statistical weight for relevant binding configurations, making them better choices for drug compounds thanlinear libraries. Simulations of a model linear and cyclic ?-AApeptide supported this idea, finding that the fluctuations of the cyclic compounds were reduced. Additionally, clustering followed by free energy calculations based on cluster populations showed that the free energy of less populated clusters rose more steeply for the cyclic than the linear. This shows that the ensemble does indeed favor lower energy configurations. Vibrational entropies obtained by quasiharmonic analysis for each cluster also showed that the entropy values within clusters was also lower for cyclic peptoids. These findings show that not only are conformers confined to fewer clusters for the cyclic peptoids, but they are more confined within clusters as well. Temperature replicaexchange of mini silk-fibrils (chapter 5) revealed the conformational fluctuations involved in spider dragline silk. These simulations showed the formation and fluctuations of secondary structure in a native-like ensemble. The stability of the ?- sheets and the residue-composition were found to best match experiment when the strands were anti-parallel within plane and parallel between planes. GGX secondary structural motifs were identified to be primarily random coils with small populations of 310-helices and 31-helices. Both left and right-handed helices were identified.The conformational fluctuations of the amorphuous region of spider dragline silk was further investigated in chapter 6, using multiple replica exchange with solute tempering (MREST), MD and pulling experiments. A higher fraction of 31-helices were observed. Additionally, the fraction of this secondary structure was increased through increasing concentration, simulation in a fiber-like environment and extension of the structure (pulling). Ultimately, the highest fraction of these helices was identified under the fiber-like conditions and extension. This indicates that this secondary structure forms during the spinning process and is favored by conversion to a fiber state.Finally, the conformational fluctuations of ligands bound to the retinoid X-receptor (chapter 7) were also investigated. Differences in water occupancy in the LBD along with shifts in the ligand binding positions suggested that these changes contribute to the binding affinity of bexarotene analogues. Differences in water occupancy between RXRalpha and RXRbeta indicated interactions that may be used to tailor compounds specific to either subtype. Finally, a hydrophobic binding pocket was identified that represent a region of potential ligand optimization to enhance the binding free energy to RXR. Summarily, my research has contributed to the understanding of conformational fluctuations and changes that occur in protein-DNA binding systems, drug-binding, regulation of chaperones via post-translations modifications and spider dragline silk.