THE EFFECTS OF HYDROPHILIC TO HYDROPHOBIC SURFACE MUTATIONS ON THE DENATURED STATE OF ISO-1-CYTOCHROME-C - INVESTIGATION OF ALIPHATIC RESIDUES

A series of hydrophilic to hydrophobic surface mutations were prepared at the highly solvent-exposed lysine 73 of iso-1-cytochrome c to asse ss the ability of such mutants to affect the energetics of the denatur ed state. In this report, the aliphatic hydrophobics (leucine, isoleuc ine, valine, alanine, glycine) were studied. The thermodynamic stabili ty of each of these mutants was determined by guanidine hydrochloride denaturation. Both the free energy of unfolding in the absence of dena turant, Delta G degrees(u)(H2O), and the slope, m, of a plot of the fr ee energy of unfolding, Delta G degrees(u), versus [guanidine hydrochl oride] show significant negative correlations with the 1-octanol to wa ter transfer free energy, Delta G(tr), of the amino acid side chain at position 73. A negative correlation with hydrophobicity is consistent with these mutants leading to more extensive hydrophobic clustering i n the denatured state, consistent with the predictions of heteropolyme r theory for compact denatured states an effect operating on the nativ e state energetics should produce a positive correlation of Delta G de grees(u)(H2O) with hydrophobicity. Infrared amide I spectroscopy indic ated native state structural perturbations for the glycine 73 and isol eucine 73 mutants. A moderate correlation of Delta G degrees(u)(H2O) w as also found with alpha-helix propensity, suggesting that both hydrop hobic effects acting on the denatured state and alpha-helix propensity are affecting the Delta G degrees(u)(H2O) values for these mutants.