Fluorescence and UV resonance Raman spectroscopy studies of HU protein binding to DNA: Protein- and sequence-induced bending

The proposed function of the bacterial DNA binding protein HU is to act primarily as an architectural protein. As an architectural protein, its function is the stabilization of DNA in a bent conformation or to induce a bend in linear DNA to allow access for other DNA binding proteins to carry out their respective functions. The overall goal of this thesis is to understand the mechanism of structural selectivity or structure-based recognition, which defines HU as an architectural protein.; The binding of HU to a linear and a bent structure of DNA have been analyzed by fluorescence and UV resonance Raman (UVRR) spectroscopy. In particular, HU binding to a 20 by bent duplex containing two phased A-tracts (A₄ -20-1) has been compared to a linear duplex containing two reverse phased A-tracts (T₄-20-1). The binding affinity for these sequences was determined at least 10-fold higher than HU binding to a random linear duplex (6.1 vs.0.68 μM−1).; Fluorescence resonance energy transfer (FRET) was used to determine the end-to-end distance of these two duplexes. Based on these measurements, it is concluded that HU induces a bend of ∼65° and stabilizes a bend even with a pre-bent substrate. From this FRET study, it is concluded that HU binding exhibits a structural specificity as shown by a 1:1 binding stoichiometry and higher binding affinity for bent DNA.; UVRR was used to probe vibrational motions of HU upon complex formation to these two duplexes. Structural differences in the protein were found upon complex formation. The amide vibrations associated with structural folds of α-helices and β sheets in HU protein become more defined in forming a complex with the A₄-20-1 than with the T₄-20-1 duplex. A decrease in intensity of the vibrational mode associated with Pro results from increased stacking interactions and intercalation of Pro with the DNA. This suggests that Pro introduces a kink that bends the linear T₄-20-1 duplex but not the A-tract duplex, A₄-20-1. These and other UVRR results support similarities between HU and IHF binding during complex formation and that the conformation of the DNA gives rise to a more structurally selective HU-DNA interaction.