Holo and apo adrenodoxin were studied by differential scanning calorimetry, absorption spectroscopy, limited proteolysis, and size-exclusion chromatography. To determine the conformational stability of adrenodoxin, a method was found that prevents the irreversible destruction of the iron-sulfur center. The approach makes use of a buffer solution that contains sodium sulfide and mercaptoethanol. The thermal transition of adrenodoxin takes place at Ttrs = 46-57 degrees C, depending on the Na2S concentration with a denaturation enthalpy of delta H = 300-380 kJ/mol. From delta H versus Ttrs a heat capacity change was determined as delta Cp = 7.5 +/- 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy (delta H = 93 +/- 14 kJ/mol at Ttrs = 37.4 +/- 3.3 degrees C) and the higher proteolytic susceptibility. The importance of the iron-sulfur cluster for the conformational stability of adrenodoxin and some conditions for refolding of the thermally denatured protein are discussed. Study holds ProTherm entries: 7246, 7247, 7248, 7249, 7250, 7251 Extra Details: Na2S(10 mM) and ascorbic acid(1 mM) were added in the experiment ferredoxin; iron-sulfur protein; protein unfolding;,scanning microcalorimetry; thermodynamics
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:33 p.m.
|Number of data points||11|
|Proteins||Adrenodoxin, mitochondrial ; Adrenodoxin, mitochondrial|
|Assays/Quantities/Protocols||Experimental Assay: Tm pH:10.5 ; Experimental Assay: dHvH pH:10.5 ; Experimental Assay: Tm pH:9.5 ; Experimental Assay: dHvH pH:9.5 ; Experimental Assay: Tm pH:8.5 ; Experimental Assay: dHvH pH:8.5 ; Experimental Assay: Tm pH:7.4 ; Experimental Assay: dHvH pH:7.4 ; Experimental Assay: Tm pH:6.5 ; Experimental Assay: dHvH pH:6.5 ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence EDKITVHFINRDGETLTTKGKIGDSLLDVVVQNNLDIDGFGACEGTLACSTCHLIFEQHIFEKLEAITDEENDMLDLAYGLTDRSRLGCQICLTKAMDNMTVRVP|