Thermal-induced dissociation and unfolding of homodimeric DsbC revealed by temperature-jump time-resolved infrared spectra.
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 D(2)O exhibit a three-state transition with the first transition midpoint temperature at 37.1 +/- 1.1 degrees C corresponding to dissociation, and the second at >74.5 degrees C corresponding to global unfolding and aggregation. The dissociation midpoint temperature of DsbC in phosphate buffer shifts to 49.2 +/- 0.7 degrees C. Temperature-jump time-resolved infrared spectra in D(2)O 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 beta-lactamase.
Li
Heng
H
Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Ke
Huimin
H
Ren
Guoping
G
Qiu
Xianggang
X
Weng
Yu-Xiang
YX
Wang
Chih-Chen
CC
D002104:Cadmium / Q000737:chemistry; D002850:Chromatography, Gel; D017666:Deuterium Oxide / Q000737:chemistry; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors; D001618:beta-Lactamases / Q000737:chemistry
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D002104:Cadmium; D001618:beta-Lactamases; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli; C526649:dsbC protein, E coli; D017666:Deuterium Oxide