Genetic fusion of subunits of a dimeric protein substantially enhances its stability and rate of folding.

Abstract

The gene V protein of bacteriophage f1 is a single-stranded DNA and RNA-binding protein composed of two identical subunits. We have constructed single-chain variants of the protein using short peptide linkers of five or six amino acids to connect the carboxyl terminus of one monomer to the amino terminus of the second monomer. The resulting subunit-fusion gene V proteins were found to bind single-stranded DNA nearly as tightly as the wild-type protein. Denaturation measurements show that the subunit-fusion gene V proteins are 5 kcal/mol (1 kcal = 4.18 kJ) more stable than the wild-type protein at a protein concentration of 10 microM. The rate of unfolding of the protein is essentially unaffected by the fusion of monomeric subunits, whereas the rate of folding is greatly enhanced. Our results suggest a simple way of obtaining a substantial thermodynamic stabilization for some oligomeric proteins.