Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro-44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule. These changes resulted in a decrease in the global stability of the protein. Further mutation of Asn-52 to Ile led to a total recovery of the wild-type stability of the sulfur-iron bond, and a partial restoration of the global stability of the protein. Substitution of Phe for His-33 did not alter the sulfur-iron bond but caused a pronounced increase in the global stability of the protein. It is suggested that this effect results from hydrophobic interaction of the Phe-33 side chain with the lower loop on the right side of the protein. Such an interaction also explains the observation that the same mutation reversed the loss of global stability caused by substitution of Val to His-26, but did not restore the strength of the sulfur-iron bond that this mutation had brought about. Study holds ProTherm entries: 9341, 9342, 9343, 9344, 9345, 9346, 9347, 9348, 9349, 9350, 9351, 9352, 9353, 9354, 9355, 9356, 9357, 9358, 9359, 9360 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:37 p.m.
|Number of data points||37|
|Proteins||Cytochrome c ; Cytochrome c, somatic|
|Assays/Quantities/Protocols||Experimental Assay: dTm ; Experimental Assay: Tm ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLFGRKTGQAPGFTYTDANKNKGITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE|