Mimicking the evolution of a thermally stable monomeric four-helix bundle by fusion of four identical single-helix peptides.

ABSTRACT

Internal symmetry is a common feature of the tertiary structures of proteins and protein domains. Probably, because the genes of homo-oligomeric proteins duplicated and fused, their evolutionary descendants are proteins with internal symmetry. To identify any advantages that cause monomeric proteins with internal symmetry to be selected evolutionarily, we characterized some of the physical properties of a recombinant protein with a sequence consisting of two tandemly fused copies of the Escherichia coli Lac repressor C-terminal alpha-helix. This polypeptide exists in solution mainly as dimer that likely maintains a four-helix bundle motif. Thermal unfolding experiments demonstrate that the protein is considerably more stable at elevated temperatures than is a homotetramer consisting of four non-covalently associated copies of a 21-residue polypeptide similar in sequence to that of the Lac repressor C-terminal alpha-helix. A tandem duplication of our helix-loop-helix polypeptide yields an even more thermally stable protein. Our results exemplify the concept that fusion of non-covalently assembled polypeptide chains leads to enhanced protein stability. Herein, we discuss how our work relates to the evolutionary selective-advantages realized when symmetrical homo-oligomers evolve into monomers. Moreover, our thermally stable single-chain four-helix bundle protein may provide a robust scaffold for development of new biomaterials.