Conformational study of intermediate in the unfolding of PcoC.

Abstract

PcoC is a small soluble protein and is considered as a kind of copper carrier in the periplasm. The PcoC protein from E. coli possesses a β-barrel fold with two metal-binding sites of Cu2+ and Cu+. In this work, different spectroscopic techniques were adopted to clarify the stability of PcoC and metals' binding property. As demonstrated in results, Ag+ and Cu2+ are capable of binding with PcoC in a proportion of 1:1. The constant for PcoC and Cu2+ was (7.27±0.21)×1013L/mol. In addition, we have explored how the cofactors affect the PcoC stability, finding that Cu2+ coordination affects both protein stability and unfolding pathway. The intermediate appeared during PcoC-Cu2+ unfolding. Further, the intermediate could be formed as CTAB interacted with PcoC. As found, the intermediate's C-terminal structure was unfolded, whereas the N-terminal was almost unaffected. Furthermore, the capability of the different unfolding degree protein with Cu2+ also indicated that the N-terminal exhibited a strong stability. Based on the anisotropy decay, tryptophan moved at a higher concentration of urea, also showing that the N-terminal was highly stable. In addition, the steered molecular dynamics simulations were performed, showing the rigidness of the N-terminal.