title: Characterization of the guanidine hydrochloride-denatured state of iso-1-cytochrome c by infrared spectroscopy.
abstract: Infrared spectroscopy has been used to monitor residual ordered structure in the denatured state of wild-type and two mutants of iso-1-cytochrome c. The technique used involves a careful digital subtraction procedure that removes spectral contributions from buffer, water vapor, and the denaturant guanidine hydrochloride. Reliable and reproducible spectra can be produced using these methods. The data for iso-1-cytochrome c show upon denaturation a shift of the structure-sensitive amide I infrared band away from the spectral region associated with random structure. Second-derivative resolution enhancement of the amide I absorption band uncovers several bands which can be associated with various residual ordered structures in the denatured state. Gradual changes in the amide I band after denaturation are also observed as the guanidine hydrochloride concentration is increased. Two single-site mutants of iso-1-cytochrome c, which have been shown to have more compact denatured states than the wild-type protein, exhibited denatured-state infrared spectra with significant differences from the wild-type protein spectra. These observations provide new insight into the characteristics of protein denatured states.
authors.affiliation: Department of Chemistry, University of Denver, Colorado 80208.
authors.forename: B E
authors.initials: BE
authors.lastname: Bowler
authors.forename: A
authors.initials: A
authors.lastname: Dong
authors.forename: W S
authors.initials: WS
authors.lastname: Caughey
mesh_terms: D000577:Amides; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry*; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013055:Spectrophotometry, Infrared; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D000577:Amides; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D006146:Guanidines; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D019791:Guanidine