The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide. Study holds ProTherm entries: 9491, 9492, 9493, 9494, 9495, 9496, 9497, 9498, 9499, 9500 Extra Details: dGH2O CuA oxidized two-state model; thermodynamic cycle; reduction potentials
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||10|
|Proteins||Cytochrome c oxidase polypeptide I+III ; Cytochrome c oxidase subunit 1|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: dG_H2O temp:75.0 C ; Experimental Assay: dG_H2O temp:20.0 C|
|Libraries||Mutations for sequence A:MAVRASEISRVYEAYPEKKATLYFLVLGFLALIVGSLFGPFQALNYGNVDAYPLLKRLLPFVQSYYQGLTLHGVLNAIVFTQLFAQAIMVYLPARELNMRPNMGLMWLSWWMAFIGLVVAALPLLANEATVLYTFYPPLKGHWAFYLGASVFVLSTWVSIYIVLDLWRRWKAANPGKVTPLVTYMAVVFWLMWFLASLGLVLEAVLFLLPWSFGLVEGVDPLVARTLFWWTGHPIVYFWLLPAYAIIYTILPKQAGGKLVSDPMARLAFLLFLLLSTPVGFHHQFADPGIDPTWKMIHSVLTLFVAVPSLMTAFTVAASLEFAGRLRGGRGLFGWIRALPWDNPAFVAPVLGLLGFIPGGAGGIVNASFTLDYVVHNTAWVPGHFHLQVASLVTLTAMGSLYWLLPNLTGKPISDAQRRLGLAVVWLWFLGMMIMAVGLHWAGLLNVPRRAYIAQVPDAYPHAAVPMVFNVLAGIVLLVALLLFIYGLFSVLLSRERKPELAEAPLPFAEVISGPEDRRLVLAMDRIGFWFAVAAILVVLAYGPTLVQLFGHLNPVPGWRLW/B:MVDEHKAHKAILAYEKGWLAFSLAMLFVFIALIAYTLATHTAGVIPAGKLERVDPTTVRQEGPWADPAQAVVQTGPNQYTVYVLAFAFGYQPNPIEVPQGAEIVFKITSPDVIHGFHVEGTNINVEVLPGEVSTVRYTFKRPGEYRIICNQYCGLGHQNMFGTIVVKE/C:EEKPKGALAVILVLTLTILVFWLGVYAVFFARG|