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 alpha-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 degrees 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 (1)H(N) (i)-(1)H(N) (i+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.
Liu
Zhijun
Z
Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People's Republic of China.
Zhang
Jiahai
J
Wang
Xingsheng
X
Ding
Yanwei
Y
Wu
Jihui
J
Shi
Yunyu
Y
D024002:HMGB Proteins / Q000737:chemistry* / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D006801:Humans; D008958:Models, Molecular; D035463:Pol1 Transcription Initiation Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics
D024002:HMGB Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; C063844:transcription factor UBF