Divergence in proteins‐ Study of Cytochrome b5 isoforms using core‐swap mutants

Abstract

Two isoforms of mammalian Cytochrome b5 exhibit remarkably different biophysical properties in spite of similar sequence and structure. The Outer mitochondrial membrane cytochrome b5 (OMb5) is presently the most stable isoform when compared to its evolutionary counterpart, the micrososmal cytochrome b5 (Mcb5). In order to understand the biophysical properties of the individual proteins, we have constructed a core swap mutant (hybb5) incorporating features from these two isoforms. The X‐ ray crystal structure, at 1.7 Å, of hybb5 has been solved and shows structural features similar to its parent proteins. The thermal stability, stability towards chaotropic agents, redox potential and heme transfer properties of hybb5 seems to have shifted in the direction of OMb5. In parallel we have performed a 10 ns Molecular dynamics simulation of OMb5, Mcb5 and hybb5. The results concur with the experimental data in that hybb5 behaves with characteristics of the OMb5 sub family. Circular Dichroism experiments of the heme free proteins (apo‐form) indicated the presence of tertiary structure. We are at present trying to further understand the apo form of hybb5 and linking it to functional divergence of OMb5 and Mcb5. The subtle structural changes that contribute to different properties in the two isoforms are being investigated via MD simulations.