Investigation of the structural stability of hUBF HMG box 5 by native-state hydrogen exchange

HMG box 5 of human upstream binding factor (hUBF) consists of three alpha-helices arranged in an L-shape with a hydrophobic core embraced by these helices and stabilized by extensive hydrophobic interactions between nonpolar residues around the core. The GdmCl-induced equilibrium unfolding transition of HMG box 5 of hUBF was monitored by both circular dichroism (CD) and fluorescence spectra. A cooperative two-state unfolding process was observed. The unfolding free energy, DeltaGU(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 +/- 0.16 kcal x mol-1 and 1.62 +/- 0.06 kcal x mol-1 x M-1, respectively. Native-state hydrogen exchange (NHX) experiments under EX2 conditions were performed. NHX results clearly show that the hydrophobic core among the three helices is a slow-exchange core. The three helices would not contribute equally to the stability of the native protein. Helix 3 appears to contribute the least to the stability. The NHX data have also allowed the local, subglobal, and global unfolding structures of hUBF HMG box 5 to be dissected, and common global and subglobal unfolding units were successfully detected.