Phenylalanine-35, which is a residue of the hydrophobic patch on the surface of cytochrome b5, has been mutated into Tyr35, His35 and Leu35 to elucidate the functions of the Phe35 and give further insight into the roles of the hydrophobic patch and/or aromatic network. The effects of these mutations on the heme environment, denaturation towards heating and the denaturant urea, redox potential and stability of protein were studied. The relative stability of cytochrome b5 and its mutants towards heating has the order Phe35Tyr > wild type > Phe35Leu > Phe35His in the oxidized state and wild type > Phe35Tyr > Phe35Leu > Phe35His in the reduced state. All the mutants exhibit decreased reduction potentials: Phe35Tyr -66 mV, Phe35His -51 mV and Phe35Leu -28 mV, which are more negative than that of the wild type. The order of redox potential reflects the relative stability in the oxidized and reduced states. A method of producing multiple mutants at a single site of a gene is also described for the first time. Study holds ProTherm entries: 3107, 3108, 3109, 3110, 3111, 3112, 3113, 14154, 14155, 14156 Extra Details: Ru(NH3)6Cl3 (0.01 mM) was added in the experiment cytochrome b5; phenylalanine; hydrophobic patch;,protein stability; redox potential
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:21 p.m.
|Number of data points||21|
|Proteins||Cytochrome b5 ; Cytochrome b5|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: dHvH temp:66.2 C, details:Additives ; Experimental Assay: ddG_H2O ; Experimental Assay: Cm ; Experimental Assay: Tm ; Experimental Assay: dHvH details:Additives Ru(NH3)6Cl3 (0.01 mM) ; Derived Quantity: dTm|
|Libraries||Mutations for sequence SKAVKYYTLEEIQKHNNSKSTWLILHYKVYDLTKFLEEHPGGEEVLREQAGGDATENFEDVGHSTDARELSKTFIIGELHPDDRSKITKPSES|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|