Destabilizing effects of replacing a surface lysine of cytochrome c with aromatic amino acids: implications for the denatured state.
A series of mutations at the highly solvent-exposed lysine 73 of iso-1-cytochrome c have been prepared by site-directed mutagenesis. These mutations were designed to probe denatured-state effects on the unfolding equilibrium of this protein. The hydrophilic amino acid Lys was replaced with the hydrophobic amino acids Met, Tyr, Phe, and Trp. The idea was to induce stabilizing hydrophobic interactions in the unfolded state, while having little effect on the folded-state energy due to the high solvent exposure of this site. Fourier transform infrared spectral analyses indicate that none of these mutations significantly affect the native fold of the protein. The stability of each protein to guanidine hydrochloride denaturation was monitored at 25 degrees C by circular dichroism spectroscopy. All four hydrophobic mutants decreased the value of delta Go uH2O, the free energy of unfolding of the protein in the absence of denaturant, by 1.0-1.5 kcal/mol. The delta Go uH2O values for these proteins correlate linearly (correlation coefficient of 0.98) with the hydrophobicity of the amino acid at position 73 of the sequence. These data are consistent with the idea that the position-73 mutants are more buried in the denatured state than in the native state, suggestive of a compact denatured state where such interactions would be possible.
Department of Chemistry, University of Denver, Colorado 80208.
B E
BE
Bowler
K
K
May
T
T
Zaragoza
P
P
York
A
A
Dong
W S
WS
Caughey
D001483:Base Sequence; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D005583:Fourier Analysis; D019791:Guanidine; D006146:Guanidines; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013816:Thermodynamics; D014162:Transfection
D003574:Cytochrome c Group; D006146:Guanidines; D019791:Guanidine; D008239:Lysine