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Thermal-Induced Dissociation and Unfolding of Homodimeric DsbC Revealed by Temperature-Jump Time-Resolved Infrared Spectra
Heng Li, Huimin Ke, Guoping Ren, Xianggang Qiu, Yu-Xiang Weng, and Chih-Chen Wang,
Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan
The thermal stability of DsbC, a homodimeric protein disulfide isomerase in prokaryotic periplasm, has been studied by using temperature-dependent Fourier transformation infrared and time-resolved infrared spectroscopy coupled with temperature-jump initiation. The infrared absorbance thermal titration curves for thermal-induced unfolding of DsbC in D2O exhibit a three-state transition with the first transition midpoint temperature at 37.1 ± 1.1°C corresponding to dissociation, and the second at >74.5°C corresponding to global unfolding and aggregation. The dissociation midpoint temperature of DsbC in phosphate buffer shifts to 49.2 ± 0.7°C. Temperature-jump time-resolved infrared spectra in D2O shows that DsbC dissociates into the corresponding germinate monomeric encounter pair with a time constant of 40 ± 10 ns independent of the protein concentration and 77% of the newly formed monomeric encounter pair undergoes further coil to helix/loop transition with a time constant of 160 ± 10 ns. The encounter pair is expected to proceed with further dissociation into monomers. The dissociation of DsbC is confirmed by size-exclusion chromatography and subunit hybridization. The in vivo oxidase activity of DsbC attributed to the monomer has also been observed by using cadmium sensitivity and the oxidative state of β-lactamase.
