Abstract
In this thesis we investigated the structure and dynamics of Myb2 protein from Trichomonas vaginalis (tvMyb2) and onconase from Rana pipiens. The Myb proteins consist of a large family of proteins with diverse functions across the biological kingdom. Most Myb proteins function as transcriptional factors by binding to DNA. Here we investigated the structure and dynamics of Myb-like transcriptional factor, tvMyb2, from Trichomonas vaginalis involved in iron-inducible transcription of ap65-1 gene. We first defined a fragment of tvMyb2 comprising residues 40–156 (tvMyb2 40–156 ) as the minimum structural unit that retains near full binding affinity with the promoter DNAs. The DNA-free tvMyb2 40–156 has a flexible and open conformation. Upon binding to the promoter DNA elements, tvMyb2 40–156 undergoes significant conformational re-arrangement and structure stabilization. Crystal structures of tvMyb2 40–156 in complex with promoter element-containing DNA oligomers showed that 5’-a/gACGAT-3’ is the specific base sequence recognized by tvMyb2 40–156, which does not fully conform to that of the Myb binding site sequence. Furthermore, Lys49, upstream of the R2 motif (52–102), also participates in specific DNA sequence recognition. Intriguingly, tvMyb2 40–156 binds to the promoter elements in an orientation opposite to that proposed in the HADDOCK model of the tvMyb1 35–141 /MRE-1-MRE-2r complex. Onconase (rONC), also known as ranpirnase or P-30 protein, initially purified from extracts of Rana pipiens oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo and is in phase III clinical trials for tumor therapy. We examined the effect of glycosylation on the structural and dynamic of onconase. The results showed that the glycan has little effect on the overall structure and dynamics of onconase. Moreover, we employed CPMG-based relaxation dispersion method and observed a novel ms time-scale protein motion elicited by nucleic acid binding. Further examination of the correlation between this collective motion and ligand binding kinetics may provide better understanding the role of dynamics in the catalytic mechanism and drug design of onconase.