Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 +/- 0.4 kcal mol(-1) to unfold the least stable substructure compared with 5.05 +/- 0.30 kcal mol(-1) for global unfolding of iso-1-cytochrome c. Thus, substructure stabilities of iso-1-cytochrome c span only approximately 1.2 kcal mol(-1) compared with approximately 8 kcal mol(-1) for horse cytochrome c. Consistent with the less cooperative folding thus expected for the horse protein, the guanidine-HCl m-values are approximately 3 kcal mol(-1)M(-1) versus approximately 4.5 kcal mol(-1)M(-1) for horse versus yeast cytochrome c. The tight free energy spacing of the yeast cytochrome c substructures suggests that its folding has more branch points than for horse cytochrome c. Studies on a variant of iso-1-cytochrome c with an H26N mutation indicate that the least and most stable substructures unfold sequentially and the two least stable substructures unfold independently as for horse cytochrome c. Thus, important aspects of the substructure architecture of horse cytochrome c, albeit compressed energetically, are preserved evolutionally in yeast iso-1-cytochrome c. Study holds ProTherm entries: 24965, 24966 Extra Details: protein folding; limited proteolysis; protein substructures; evolutionary conservation; folding cooperativity; alkaline transition
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:55 p.m.
|Number of data points||7|
|Proteins||Cytochrome c iso-1 ; Cytochrome c iso-1|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence TEFKAGSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTDANIKKNVLWDENNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACE|