A lysine 73-->histidine variant of yeast iso-1-cytochrome c: evidence for a native-like intermediate in the unfolding pathway and implications for m value effects.
In this paper we report thermodynamic studies on a variant of yeast iso-1-cytochrome c in which a surface lysine residue at position 73 has been replaced with a histidine (H73). Guanidine hydrochloride denaturation studies monitored by circular dichroism spectroscopy indicated decreased thermodynamic stability (a lower delta G(o)(u)H20) and a smaller m value for the H73 protein as compared to the wild type (WT) protein. Further investigations to probe the causes for the thermodynamic stability differences between the two proteins involved guanidine hydrochloride and urea denaturations monitored by tryptophan fluorescence. The stability of heme ligation in the denatured state in the presence of either guanidine hydrochloride or urea was monitored by the spin-state transition of the heme iron induced by pH. None of these studies supported the hypothesis that the decreased m value was due to heme-His73 ligation in the denatured state. Guanidine hydrochloride denaturations monitored by the change in the extinction coefficient at 695 nm, which is sensitive to the presence of heme-Met80 ligation, revealed a native-like intermediate for the H73 protein, probably caused by displacement of the Met80 heme ligand by histidine 73 at guanidine hydrochloride concentrations much lower than required for full cooperative unfolding. Presence of the native-like intermediate is most likely the cause of the smaller m value and decreased thermodynamic stability for the CD-monitored H73 protein unfolding as compared to the unfolding of the WT protein. Guanidine hydrochloride denaturations in the presence of 200 mM imidazole provide further evidence in support of the proposed mechanism.
Godbole
S
S
Department of Chemistry and Biochemistry, University of Denver, Colorado 80208, USA.
Dong
A
A
Garbin
K
K
Bowler
B E
BE
D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000235:genetics*; D045304:Cytochromes c; D005453:Fluorescence; D005656:Fungal Proteins / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D005656:Fungal Proteins; D006146:Guanidines; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan; D014508:Urea; D045304:Cytochromes c; D019791:Guanidine