Amino acid replacements in a homodimeric protein, Streptomyces subtilisin inhibitor, at the position of Val13, which is located at the center of the beta-sheet interface of the domain-domain interaction, changed both the overall stability and the denaturational scheme. All the mutant forms lost stability, losses in free energy at 82.21 degrees C at pH 7.0 obtained by calorimetric measurements ranging from 10.3 kcal (mol dimer)-1 for the Gly mutant, which involved a loss in enthalpy without a compensating loss in entropy, to 0.84 kcal (mol dimer)-1 for the Ile mutant, which involved comparable losses in enthalpy and entropy. A gain in enthalpy for the Ala mutant was overcome by a gain in entropy, resulting in a loss in free energy by 6.78 kcal (mol dimer)-1. It was found that decreases in enthalpy and entropy showed good correlation with increasing side-chain hydrophobicity, while no such correlation was present for free energy. Replacements with Gly, Ala, Met and Phe altered the thermal denaturational scheme from N2 reversible 2D, which was the case for the Leu and Ile mutants as well as for the wild-type, to N2 reversible 2N* reversible 2D, where the first step can be regarded as a dissociation of the native dimer followed by a major unfolding in the second step. The free energy acquired by forming the dimer was estimated to be 2.03 kcal mol-1 for the Ala mutant at 66.95 degrees C at pH 9.5 and lower for the other three mutants. Study holds ProTherm entries: 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 14044, 14045, 14046, 14047, 14048, 14049, 14050, 14051, 14052, 14053, 14054, 14055 Extra Details: ddG was computed at Tm of wild type calorimetry; protein stability; hydrophobicity; mutagenesis;,domain-protein stability; domain interaction
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:19 p.m.
|Number of data points||60|
|Proteins||Subtilisin inhibitor ; STREPTOMYCES SUBTILISIN INHIBITOR|
|Assays/Quantities/Protocols||Experimental Assay: dG buffers:glycine: 25 mM, pH:9.5 ; Experimental Assay: dG buffers:phosphate: 25 mM, pH:7.0 ; Experimental Assay: dHcal buffers:glycine: 25 mM, pH:9.5 ; Experimental Assay: Tm buffers:glycine: 25 mM, pH:9.5 ; Experimental Assay: dHcal buffers:phosphate: 25 mM, pH:7.0 ; Experimental Assay: Tm buffers:phosphate: 25 mM, pH:7.0 ; Derived Quantity: ddG buffers:glycine: 25 mM, pH:9.5 ; Derived Quantity: ddG buffers:phosphate: 25 mM, pH:7.0 ; Derived Quantity: dTm buffers:glycine: 25 mM, pH:9.5 ; Derived Quantity: dTm buffers:phosphate: 25 mM, pH:7.0|
|Libraries||Mutations for sequence DAPSALYAPSALVLTVGKGVSATTAAPERAVTLTCAPGPSGTHPAAGSACADLAAVGGDLNALTRGEDVMCPMVYDPVLLTVDGVWQGKRVSYERVFSNECEMNAHGSSVFAF|