Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

Legacy Department


Committee Member

Brian Dominy, Committee Chair

Committee Member

Steven Stuart

Committee Member

Julia Brumaghim

Committee Member

Weiguo Cao


This thesis employed molecular dynamics (MD) simulations to investigate the effects of substrate binding and site-direct mutation of proteins. Chapter 2 performed MD simulations to examine the substrate effects on the behavior of Atm1 type ABC exporter. We found that thiol-containing glutathione (GSH) bound Atm1 type ABC exporter undergoes the largest conformational changes characterizing the nucleotide binding domain (NBD) coming closer together. The intracellular loops ICL1 and ICL2 between NBDs and transmembrane domains (TMDs) associated with the biggest fluctuation in the first principle component from PCA analysis in GSH bound system. Also, unlocking and slide along each other of two C-helices were observed in GSH system. The orientation of GSSG and GSH are not sterically constrained by the membrane protein due to its rotation within the large transmembrane hole. Therefore, it is impossible to assign specific binding sites in the transmembrane hole to either substrate. The following chapter investigated the allosteric communication of both the wild-type and the double mutation Y195F/Q272A of the Atm1 type ABC transporter by MD simulations and compared to two single mutant Y195F and Q272A of Atm1 type ABC exporter in detail. In the 200 ns MD simulations, we observed that both double mutation Y195F/Q272A and single mutation Y195F exhibited large conformational flexibility using RMSD analysis. A ‘semi-closed' geometry detected in Y195F/Q272A MD trajectory that is closer than the starting crystal structure suggests that the two NBDs approached each other. The single mutation Q272A fail to the link the TM4 and TM6, which in turn exhibits insensitive ATPase hyperactivity due to the loose contact with the nearby residues with no bulky sidechain. Finally, further PCA indicated that the two NBDs rotate in opposite directions, which results in the asymmetric NBD dimerization. This in turn, leads to the allosteric communication between TMDs and NBDs. In chapter 4, questions including the physicochemical mechanisms motivating the evolution of Tth UDGa are tackled through density function theory (DFT) methods, MD simulations, and a successive analysis of thermodynamic properties associated with the enzyme activity using MD trajectories. Transition states have been successfully located from DFT frequency and IRC calculations based on the B3LYP/6-31+G(d) level of theory. TthUDGa bound reactant in a typical 3'-exo sugar ring conformation that favors oxacarbenium ion through nucleotide backbone distortion. An encouraged Spearman correlation (rs=0.786, r2=0.554) was achieved by comparison to the experimental data. These results suggest that the electrostatic interaction changes appear to be a major role in enzyme efficiency resulting from mutation. A detailed analysis of the transition state conformation of the glycosidic bond scission, binding affinity between TthUDGa protein and flipping out uridine, and catalytic activity of TthUDGa/DNA complexes suggests that the transition state stabilization might be contributing factors for the evolutionary optimization of TthUDGa/DNA complexes.