This is a new proposal aimed at obtaining information on the structural, thermodynamic, and dynamic aspects of non-sequence specific modes of protein-nucleic acid recognition by the HMG-box motif. The objective will be to understand the recognition in terms of two limiting mechanisms: recognition of preformed structure (lock and key) vs induction of structure in inherently flexible DNA sequences (induced fit). A multifaceted approach is proposed in order to give the most complete picture: electrophoretic mobility shift assays and radical footprinting for characterizing binding of different DNA sequences, isothermal titration calorimetry for determining thermodynamic parameters, EPR for determining DNA dynamics, and NMR for structure analysis of the proteins and its complexes with DNA. The goal is to characterize the binding of nucleic acids containing one each of ~7 structural and/or dynamical features to a single HMG-box from the Xenopus Laevis Upstream Binding Factor (xUBFBox1).
