Investigation of the Structural Stability of hUBF HMG Box 5 by Native-State Hydrogen Exchange

• Published in: Biochemistry (Easton) 2007-02, Vol.46 (5), p.1293-1302
• Main Authors: Wang, Dandan, Zhang, Jiahai, Jin, Xianju, Wu, Jihui, Shi, Yunyu
• Format: Article
• Language: English
• Subjects: Circular Dichroism; Deuterium Exchange Measurement; HMGB Proteins - chemistry; Pol1 Transcription Initiation Complex Proteins - chemistry; Protein Denaturation; Spectrometry, Fluorescence; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061682r

HMG box 5 of human upstream binding factor (hUBF) consists of three α-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, ΔG U(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 ± 0.16 kcal·mol-1 and 1.62 ± 0.06 kcal·mol-1·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.