Temperature-induced partially unfolded state of hUBF HMG Box-5: conformational and dynamic investigations of the Box-5 thermal intermediate ensemble.

Human upstream binding factor (hUBF) HMG Box-5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA-binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α-helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three-state thermal unfolding equilibrium of hUBF HMG Box-5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box-5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box-5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box-5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential 1HNi-1HNi+1 NOE analyses indicate that helices 1 and 2 are native-like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix-turn-helix folding model of Box-5, where helices 1 and 2 potentially form the helix 1-turn-helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley-Liss, Inc.