Protein interactions leading to conformational changes monitored by limited proteolysis: Apo form and fragments of horse cytochrome c

Proteolysis experiments have been used to monitor the conformational transi tions from an unfolded to a folded state occurring when the apo form of hor se cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic pr obe, in view of the fact that the broad substrate specificity of this prote ase allows digestion at many sites along a polypeptide chain. The rather un folded apo form of cyt c binds heme with a concomitant conformational trans ition to a folded species characterized by an enhanced content of helical s econdary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalen t complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, i s rather resistant to proteolysis, while the individual fragments are easil y digested. Fragment 57-104 is fast degraded to several peptides, while fra gment 1-56 is slowly degraded stepwise from its C-terminal end, leading ini tially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysi s, fragments 1-38, 1-35, 1-33. and 1-31. Thus, proteolysis data indicate th at the heme containing fragment 1-56 has a rather compact core and a C-term inal flexible tail. Upon prolonged incubation of the complex of fragments 1 -56 and 57-104 (nicked cyt c) with proteinase K. a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex ma tches the omega -loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well wi th the extent of folding of the protein substrates, as deduced from circula r dichroism measurements. Thus, our results underscore the utility of prote olytic probes for analyzing conformational and dynamic features of proteins . Finally, a specific interest of the cyt c fragment system herewith invest igated resides in the fact that the fragments are exactly the exon products of the cyt c gene.