Insight into Structure and Function of EcoRI Through Detailed Characterization of Nitroxide Side Chains Using Molecular Dynamics Simulations

This thesis describes molecular dynamics simulations used in an effort to investigate the orientation and dynamics of the methane thiosulfonate spin label (MTSSL) on solvent exposed sites of the loop (R131C) and beta strand (S180C) of the specific EcoRI-DNA complex. Also, the results are compared with those of a well-studied solvent exposed alpha helical site (SEHS). By modeling the spin label at these sites using the all-atom simulation, we intend to explain the observed double electron-electron resonance (DEER) distance distribution data of the specific complex of R131C and S180C mutants. EcoRI is a restriction endonuclease, which binds and cleaves the specific DNA sequence 5'GAATTC3'. The arms of EcoRI are thought to play an important role in its high binding affinity. However, the exact reason(s) why it shows such an extraordinary binding specificity is still being investigated. Therefore, to shed light on the high binding specificity of the enzyme, distance measurements were previously obtained using DEER, providing the structural constraints of the arms of EcoRI. Since the DEER experiment provides inter-spin label distances, simulations can be used to de-convolute the spin label contribution to extract the inter-atomic distances (C-C) of the arms of EcoRI. In order to accomplish this, we intend to effectively reproduce the experimental DEER data using molecular dynamics simulations. Utilizing simulations, the structural constraints contained in experimental data can be obtained and compared with the X-ray crystal structure of the specific EcoRI-DNA complex. Although we attempt to explain the DEER data by examining spin-label conformations, the study also involves linking the dynamics of the spin label to the MD-generated distance distributions