Effects of natural mutations (L94I and L94V) on the stability and mechanism of folding of horse cytochrome c: A combined in vitro and molecular dynamics simulations approach

Abstract Known crystal structures of 10 cytochromes (cyts) c from different sources led to the conclusion that natural mutations in these proteins does not affect their 3D structure, hence evolution preserved structure for function. A sequence alignment of horse cyt c with all other 284 cyts c led to two important conclusions: (i) Leu at position 94 is conserved in all 30 mammalian known sequences, and (ii) there are 14 other species which have either Val or Ile at 94th position. We asked a question: Is the avoidance of substitution by Val or Ile at position 94 in the mammalian cyts c by design or by chance? To answer this question, we introduced natural substitutes of Leu94 by Val and Ile in horse cyt c using site-directed mutagenesis. Here, from our in vitro and molecular dynamic simulation studies on L94V and L94I mutants, we concluded that (i) although the natural mutations destabilize the wild type cyt c, it does not significantly affect the mechanism of folding of the protein, (ii) urea-induced denaturation of WT cyt c and its mutants is a two-state process, and (iii) denaturation of WT cyt c and its mutants by guanidinium chloride is not a two-state process.