UC Irvine

Solution state NMR was used to study two DNA-binding proteins. Chapter one covers the different NMR techniques used, including hydrogen exchange, relaxation, chemical shift analysis, and temperature coefficients. In addition, I also implement circular dichroism and protein sequence analysis to supplement the NMR studies. Chapter two covers the p53 project, in which we compare protein dynamics between WT and rescue mutants to understand the mechanism of stabilization. We measured and compared dynamics from NMR hydrogen exchange rates of backbone amides and find that both rescue mutants impose a global stabilizing effect that dampens their motions in the beta-sandwich compared to WT DBD. We also compared NMR relaxation results to obtain flexibility information in the ps to ns timescale regime. Chapter three covers the dynamics of the DinB homolog polymerase (Dbh). This polymerase belongs to the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning in a wide range of temperatures. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2-65°C) and metal binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbation analysis with and without magnesium and manganese. HX reveals that both the thumb and finger domains are very dynamic relative to the palm and LF domains. These trends remain true at high temperatures with dynamics increasing as temperatures increase from 35 °C to 50 °C. Notably, NMR and CD spectra show that Dbh tertiary structure cold denatures beginning at 25 °C and increasing in denaturation as the temperature is lowered to 5 °C with little change observed by CD. Chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, even in the absence of DNA.