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FOR THE RECORD

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

¹ Department of Biology, Yeshiva University, New York, New York 10033, USA
² Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, USA

Reprint requests to: Michael Mossing, Department of Chemistry and Biochemistry, University of Mississippi, University, MS 38677, USA; e-mail: mmossing{at}olemiss.edu; fax: (662) 915-7300.

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 ß-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 ß-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small.

Keywords: Cro repressor; ß hairpin; circular dichroism; protein stability; engineered monomer

Abbreviations: DG, Cro K56[DGEVK] • GG, Cro K56[GGEVK] • GT, Cro K56[GTEVK] • CD, circular dichroism • GdnHCl, guanidine hydrochloride

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