title: Investigation of the structural stability of hUBF HMG box 5 by native-state hydrogen exchange.
abstract: 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.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, Peoples Republic of China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Wang
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhang
authors.forename: Xianju
authors.initials: X
authors.lastname: Jin
authors.forename: Jihui
authors.initials: J
authors.lastname: Wu
authors.forename: Yunyu
authors.initials: Y
authors.lastname: Shi
mesh_terms: D002942:Circular Dichroism; D041961:Deuterium Exchange Measurement; D024002:HMGB Proteins / Q000737:chemistry*; D035463:Pol1 Transcription Initiation Complex Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D024002:HMGB Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; C063844:transcription factor UBF