Conferment of folding ability to a naturally unfolded apocytochrome c through introduction of hydrophobic amino acid residues.

Hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic Pseudomonas aeruginosa cytochrome c(551) (PA c(551)), which is structurally homologous to AA c(555) in the holo state. Here we hypothesized that the exceptional apo AA c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a PA c(551) variant (denoted as PA-nh) carrying the nine hydrophobic residues at structurally corresponding positions. Circular dichroism experiments showed that the apo PA-nh variant became folded, unlike the wild-type apo PA c(551), and exhibited much higher stability than the wild type. Another difference between the holo forms of AA c(555) and PA c(551) is the existence of an extra helix in the former. Introduction of the amino acid residues forming the extra helix of AA c(555) into the PA-nh variant did not significantly affect its folding ability in the apo state. Therefore, the nine hydrophobic residues introduced into the apo PA-nh variant were enough to confer the folding ability. PA c(551) represents the first example of the conversion of an intrinsically unfolded apocytochrome c into an autonomously folded one, which was revealed by means of a protein engineering method without heme. Although heme is generally considered to be a trigger of apocytochrome c folding, the present results demonstrate a new heme-independent folding mechanism.