Stability of monomeric Cro variants: Isoenergetic transformation of a type I to a type II -hairpin by single amino acid replacements

Abstract

The thermodynamic stabilities of three monomeric variants of the bacteriophage Cro repressor that differ only in the sequence of two amino acids at the apex of an engineered -hairpin have been determined. The sequences of the turns are EVK-XX-EVK, where the two central residues are DG, GG, and GT, respectively. Standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins. Thermal denaturation yields van’t Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58°C. Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations. As predicted from statistical surveys of amino acid replacements in -hairpins, energetic barriers to transformation from a type I turn (DG) to a type II turn (GT) can be quite small.