Structural studies of XPC binding domain of hHR23B by NMR and CD

Abstract

XPC is considered to be the first protein to recognize damaged DNA in global genome nucletide excision repair and the activity is greatly stimulated by addition of hHR23B protein. From the domain study, residues from 277 to 332 were enough to stimulate XPC activity. Structural studies of XPC binding domain of hHR23B could identify the structural changes in XPC bound complex, thus explain the enhanced damage recognition activity upon complex formation. To address this question, the expression vector of XPC binding domain of hHR23B (277 332) was constructed, and the domain was purified. CD experiments of XPC binding domain of hHR23B showed that its secondary structure was mainly composed of alpha helical forms, and strong prediction was made that this helices contains amphipathic features. However, this helical, amphipathic XPC binding domain of hHR23B suffered from severe aggregation when it was concentrated for NMR studies, leading to broad, uninterpretable spectra. For structural studies using NMR spectroscopy, the optimization for the best soluble condition was urgently required. The proper detergent and its amount for high protein concentration without structural modification of the protein were screened using near and far UV CD spectra and NMR methods. The secondary structure of XPC binding domain of hHR23B was not sensitive to pH, salt, organic solvent such as methanol and trifluoroethanol, and various detergents, which includes sodium dodecyl sulfate, dodecyl phosphate choline and octylglucoside. The protein was effectively concentrated up to 0.6 mM in 15 % methanol, and over 1 mM in 40mM octyl glucoside. Although 40 mM octyl glucoside was the most effective solubilizer, some distinct changes of chemical shifts were observed in $^{15}N-HSQC$ spectrum. But regarding to the near UV CD experiment, these chemical shift change may result from binding of octylglucoside to the protein and the structure being sustained. However, $^{15}N-HSQC$ spectra in the ...