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CopC is a periplasmic copper Chaperone protein that has a b-barrel fold and two metalbinding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site-directed mutagenesis. The far-UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the protein's function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The D obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ=mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the induced by urea for Y79WW83L have a loss of 11.44 kJ=mol, but for Y79WW83F did a raise of 1.82 kJ=mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC.

The CopC protein from pseudomonas syringae pathovar tomato is expressed as one of four proteins encoded by the operon CopABCD that is responsible for copper resistance. And there are one tryptophan (83), one tyrosine (79), and three phenylalanines (35, 43, 99) in apoCopC. The fluorescence peak of apoCopC is located near 320 nm, and the peak shifts toward 353 nm in the presence of 10 mol·L −1 urea with excitation at 280 nm. Using urea as a chemical agent, the conformational stabilities of apoCopC and were monitored by fluorescence spectrum in 20 mmol·L −1 phosphate buffer and 100 mmol·L −1 sodium chloride at pH 6.0. The free energy of stabilization for apoCopC and is 16.29±0.65 kJ·mol −1 and 26.26±0.35 kJ·mol −1 , respectively. The distance between the tryptophan residue and the Cu 2+ in has been studied by observing Förster type nonradiative energy transfer. And it is calculated to be 11.6 Å.

The interaction between mercuric ion and apoCopC in the absence or presence of cupric ion was investigated through difference UV spectra in Hepes buffer (10 mmol•L -1 ) at pH 7.

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In the current three-state protein unfolding model, the two transitions are considered to be independent and each transition is fitted to a two-state unfolding model. This three-state unfolding process is therefore composed of two sequential two-state unfolding processes. In this paper, a modified method is presented to determine the value of the unfolding free energy [ΔG 0 total (H 2 O)] for the three-state unfolding equilibrium of proteins. This method is demonstrated on the apoCopC protein mutant, Y79W-W83F-Cu, which unfolds via a three-state process. The value of ΔG 0 total (H 2 O) calculated using the modified method was found to be more accurate in determining ΔG 0 total (H 2 O) than the previously reported method.protein stability, three-state model, Y79W-W83F, unfolding Citation: Zheng X Y, Yang B S. An improved method for measuring the stability of a three-state unfolding protein.

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