Guanidine hydrochloride induced equilibrium unfolding of mutant forms of iso-1-cytochrome c with replacement of proline-71.
Proline-71, an evolutionally conserved residue that separates two short alpha-helical regions, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, J. F., Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F. (1985) J. Biol. Chem. 260, 13225-13236]. Treatment of these proteins with a specific sulfhydryl blocking reagent (methyl methanethiosulfonate) to block Cys-102 has allowed investigation of the properties of monomeric forms of the proteins, denoted iso-1-MS. Comparison of the UV-visible absorbance properties (pH 6, 20 degrees C) shows minor differences between the normal Pro-71 iso-1-MS and two of the three mutant proteins. The Val-71 iso-1-MS protein has absorbance properties indistinguishable from those of the normal Pro-71 iso-1-MS protein, but the Ile-71 iso-1-MS and Thr-71 iso-1-MS proteins show reduced intensity of the 695-nm absorbance band and a small shift in the Soret maximum, from 408 nm for the Pro-71 iso-1-MS and Val-71 iso-1-MS proteins to 406 nm for the Thr-71 iso-1-MS and Ile-71 iso-1-MS proteins. Second derivative spectroscopy is used to assess differences in the polarity of the environment of tyrosine residues. The average degree of exposure of tyrosines to solvent is similar in all four proteins: 0.39 for the normal Pro-71 iso-1-MS and Val-71 iso-1-MS proteins; 0.40 for the Ile-71 iso-1-MS protein.(ABSTRACT TRUNCATED AT 250 WORDS)
D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D009154:Mutation; D011392:Proline; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D006146:Guanidines; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D011392:Proline; D019791:Guanidine