Structural dynamics of proteins in functional control

Abstract

Manipulation of protein conformational ensembles are well-known phenomenon yielding the development of rational drug design strategies. As a novel contribution, the aim of this thesis is to mechanistically identify and perturb the key sites that would control and modify the ensemble of conformations and to investigate the pos sible recovery effects of these perturbations which could result in finding allosteric binding sites. Ras-related C3 botulinum toxin substrate 1 (Rac1) has been known for important regulatory roles in signaling pathways, and abnormal activity of Rac1 due to hot spot mutations promotes cancer progression, such as P29S mutation which involves in melanoma. For the purpose of exploring the dynamic mechanism of the conformational shift due to mutations so that drugs mimicking suppressor mutations might be designed to rescue the effect of the oncogenic mutations, Rac1 and P29S was chosen as exemplary cases. The rescue positions at/in close vicinity to the mechanisti cally informative regions are hypothesized to have the capacity to alter the dynamics related to protein’s function. In silico mutations are introduced as perturbations to the hinge residues of P29S mutant, and their dynamic response effects are analyzed by Molecular Dynamics (MD) simulations. The results indicate wild type and P29S mutant are consisted of substates that are distinguished by the conformations of the residues that maintain Mg2+ coordination. Moreover, altered dynamics of functional regions in P29S mutant are important in adopting fast cycling property by increasing the binding affinity towards downstream effectors along with the possible decreased binding ability to upstream effectors. Lastly, C6A, L19A, T75A and E148A mutations could be rescue mutations as they begin to restore the dynamic behavior of GTP and upstream/downstream effector binding residues.