The Stability Of CopC And The Role In Copper Regulation

The CopC protein has been proposed to be a copper chaperone protein consisting of 102 amino acids. NMR and EXAFS structural data revealed a β-barrel topology and two copper binding sites, separated by ～30 A. One site is specific for Cu+ while the second is specific for Cu2+. The ligand environments are Cu+(His)(Met)x (x=2 or 3) and Cu2+(His)2(Asp)(Glu)(OH2). Three of a total of four His residues are proposed as ligands (H48 for Cu+; H1, H91 for Cu2+) as are at least two of the four Met residues M40, M43, M46, and M51.Aromatic side chains such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) have a unique role in the protein folding. Given that these aromatic side chains are among the most hydrophobic, they are important in hydrophobic clustering. Furthermore, Trp, Phe, and Tyr may be conserved to ensure the structural stability of folding. In addition, the aromatic amino acid Trp is the intrinsic fluorescent probe most widely used for monitoring protein conformational changes, dynamics, and intermolecular interactions due to its indole chromophore, which is very sensitive to its environment. The mutant of CopC was obtained by site directed mutangenesis. Recombinant plasmids were transformed in E.coli BL21, and then the expressions of mutant were performed by the induction of IPTG, then those mutants were purified by anion and cation exchange chromatography. The final products were examined by SDS-PAGE, Mass spectrum, UV-Visual spectra, and all of the protein products were of high purity.The mechanism by which proteins fold to their unique native conformations from an initially disorganized form is one of the fundamental problems in molecular biology. In the study of proteins fold or refold standard Gibbs free energy, which is the single most important parameter for quantitating protein stability and comparing stabilities of closely related proteins. In order to exactly calculate unfolding free energy values of three-state models, the new model is present. In this model, it is considered that proteins are considered to be composed of structural elements. The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method. The measured standard Gibbs free energy is the sum of the free energy changes for each structural element. In addition, the molecular simulation was used to investigate the mechanism of protein unfolding.Hydrogen bonding and hydrophobic effect are essential to the structure and stability of globular proteins. A common but unique feature of most Greek key proteins is the"tyrosine corner." As a consequence of its high conservation, the tyrosine corner has been postulated to be important in the folding and stabilization of native protein. In order to explore the roles of Trp83 and Tyr79 on protein stability, this study reports on guanidinum ion (GdnHCl), as well as urea unfolding studies of apoCopC and its mutations monitored by fluorescence spectroscopy. The results suggest that the hydrogen bond interactions between Tyr79 and Thr75 as well as the interactions between the aromatic ring of Trp83 and other residues have an important role in stabilizing apoCopC. The differences in microenvironment around the two important resides are interpreted based on the quenching experiments and fluorescence lifetime measurement results. These findings demonstrate that the microenvironment around Trp83 is more hydrophobic than that around Tyr79 in apoCopC.The nature and role of intermediates had been the subject of much heated debate in the field of protein folding. Historically, intermediates were viewed as essential stepping-stones that guide a protein through the folding process to the native state. Commonly, the intermediate was obtained by the interaction of ionic surfactants with proteins, which possessing native-like secondary structure elements but lacking the tight packed tertiary structure of the native state. We report studies of the interaction of apoCopC with the anionic surfactant SDS, CTAB, urea, and guanidinium hydrochloride (GdnHCl), using fluorescence spectroscopy of the intrinsic Trp, far-UV circular dichroism (CD) spectroscopy,2-p-toluidinylnaphthalene (TNS) fluorescence, fluorescence lifetime, and fluorescence anisotropy measurements, and steered molecular dynamics (SMD) simulations to characterize the structure of the intermediate in the presence of surfactant solution. Further, the effects of metals on the stability of the intermediate have been investigated. The interaction of SDS with CopC resulted in the formation of a partially folded intermediate. In this intermediate, the structure of the C-terminal is unfolded, whereas the N-terminal retains the native structure. The interaction of CTAB with CopC has been found to stabilize a second partially folded intermediate, which is considered that the structure of C-terminal was unfolded completely, whereas the structure of N-terminal was unfolded partially.In order to investigate the effect of small molecular on the mechanism of copper transfer, the interaction between HSSC and apoCopC has been studied in detail by means of UV, fluorescence and fluorescence life time measurement. The results suggested that HSSC can forma novel supra molecular system with apoCopC, which can form a 1:1 host-guest inclusion supra molecular complex with HSSC, In addition, the stoichiometric ratio of Cu2+ and HSSC was 1:1. Furthermore, it was also found that HSSC quench the fluorescence of apoCopC by the static quenching process. The formation of apoCopC-HSSC complex depended on the cooperation of the van der Waals force and hydrogen bond, and the binding average distance between apoCopC and HSSC was determined. What is more, the binding site of HSSC to apoCopC was shown vividly by an automated public domain software package ArgusLab 4.0.1.It confirms the presence of two separate but interdependent binding sites, one with high specific affinity for Cu+ and the other for Cu2+. The structural lability of the two sites is revealed for the fully loaded Cu+ Cu2+ form. If the Cu+ site only is occupied in solution, the Cu ion is oxidized rapidly by dioxygen. However, if the Cu2+ site is also occupied, the Cu+ site is stable. The availability of an unoccupied site of higher affinity in wild-type or variant forms induces intermolecular transfer of either Cu+ or Cu2+ while buffering free cupric ion concentrations at sub-picomolar levels. In order to investigate the copper transfer mechanism, the reduced rate was measured by fluorescence spectra.