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 assess the ability of such mutants to affect the energetics of the denatured state. In this report, the aliphatic hydrophobics (leucine, isoleucine, valine, alanine, glycine) were studied. The thermodynamic stability of each of these mutants was determined by guanidine hydrochloride denaturation. Both the free energy of unfolding in the absence of denaturant, delta GouH2O, and the slope, m, of a plot of the free energy of unfolding, delta Gou, versus [guanidine hydrochloride] show significant negative correlations with the 1-octanol to water transfer free energy, delta Gtr, 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 in the denatured state, consistent with the predictions of heteropolymer theory for compact denatured states; an effect operating on the native state energetics should produce a positive correlation of delta GouH2O with hydrophobicity. Infrared amide I spectroscopy indicated native state structural perturbations for the glycine 73 and isoleucine 73 mutants. A moderate correlation of delta GouH2O was also found with alpha-helix propensity, suggesting that both hydrophobic effects acting on the denatured state and alpha-helix propensity are affecting the delta GouH2O values for these mutants.
Herrmann
L
L
Department of Chemistry, University of Denver, Colorado 80208.
Bowler
B E
BE
Dong
A
A
Caughey
W S
WS
D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics*; D045304:Cytochromes c; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
D000596:Amino Acids; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c