Thermal-Induced Dissociation and Unfolding of Homodimeric DsbC Revealed by Temperature-Jump Time-Resolved Infrared Spectra
AUTOR(ES)
Li, Heng
FONTE
The Biophysical Society
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2776246Documentos Relacionados
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