Refolding a disulfide dimer of cytochrome c

Bryant, Christopher S.; Strottmann, James M.; Stellwagen, Earle

doi:10.1021/bi00335a011

Cited by 20 publications

(10 citation statements)

References 16 publications

(23 reference statements)

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“…The results of these studies on the acetamide-derivatized monomer of iso-1 cytochrome c clearly show that protein conformations intermediate between monomer and dimer forms are possible, depending on the polarity and charge of the modifying group. Such a conclusion is consistent with results of protease digestion and denaturation studies of Montonaga et al (1965b) and Bryant et al (1985) performed on the sulfiteand iodoacetate-derivatized monomer of iso-1 cytochrome c. More detailed analyses of the structural alterations induced by Cys-102 modification of iso-1 ferricytochrome c must await characterization of the protein derivatives by X-ray diffraction or multidimensional NMR methods.…”

Section: Discussionsupporting

confidence: 78%

“…3). Studies at higher temperatures were not pursued because Bryant et al (1985) have reported that the heat denaturation midpoint temperature of the disulfide dimer of iso-1 ferricytochrome c is 316 K.…”

Section: Temperature Dependence Of Selected Hyperfine-shifted Proton mentioning

confidence: 99%

“…The amino acid situated next to the carboxy-terminal amino acid in iso-1 cytochrome c is a cysteine (Cys-102), while this position is occupied by an alanine in iso-2 cytochrome c and a threonine residue resides at position 102 in horse cytochrome c. The presence of the cysteine residue in iso-1 cytochrome c leads to the formation of variable amounts of disulfidelinked dimers of yeast iso-1 cytochrome c upon oxidation (Montonaga et al, 1965a), which is contrary to what happens in most other proteins having free sulfhydryl groups. Questions concerning the possibility of a functional role for the unusually reactive Cys-102 have lead to studies of various types of Cys-102 chemically modified forms of iso-1 cytochrome c which have been performed in a number of laboratories using such reagents as sodium sulfite (Montonaga et al, 1965a), iodoacetamide (Zuniga and Nall, 1983), iodoacetate (Bryant et al, 1985), and 5,5' dithiobis (2-nitrobenzoate) (DTNB) (Birchmeirer et al, 1976). The results of those studies revealed that the sulfite-and iodacetate-modified and dimer forms of iso-1 cytochrome c were much more susceptible to protease digestion and denaturation than the native monomeric form of the protein and suggested that modification of the free SH group (either by dimerization or via specific reagents) resulted in 3Abbreviations: SDS, sodium dodecyl sulfate; DTNB, 5,5' dithiobis (2-nitrobenzoate); SH, sulfhydryl; AM, acetamide; TNB, thionitrobenzoate; SCH3, methanethio group; iso-l-SH, native (Cys-102 unmodified) monomeric yeast iso-1 cytochrome c; iso-I-SAM, Cys-102 acetamide-derivatized monomer of yeast iso-1 cytochrome c; iso-I-STNB, Cys-102 thionitrobenzoate-derivatized monomer of yeast iso-1 cytochrome c; iso-I-SSCH3, Cys-102 S-methylated derivatized monomer of yeast iso-1 cytochrome c; E,,.…”

Section: Introductionmentioning

confidence: 99%

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A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

Moench¹,

Satterlee²

1995

J Protein Chem

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Derivatives of yeast iso-1 cytochrome c, chemically modified at Cys-102 (Cys-102 acetamide-derivatized monomer, Cys-102 thionitrobenzoate-derivatized monomer, Cys-102 S-methylated monomer, and the disulfide dimer), exhibit different spectral and physicochemical properties relative to the native, unmodified protein, depending on the nature of the modifying group. The results of proton NMR studies on the Cys-102 acetamide-derivatized monomer of iso-1 ferricytochrome c indicate that the conformational characteristics of the heme environment in this protein derivative are intermediate between those of the unmodified monomer and disulfide dimer forms of the protein. Measurements of the pKa of the alkaline transitions of the five forms of iso-1 ferricytochrome c provided values of 8.89, 8.82, 8.67, 8.47, and 8.50 for the unmodified monomer, S-methylated monomer, acetamide-derivatized monomer, thionitrobenzoate-derivatized monomer, and disulfide dimer, respectively. The results of proton NMR studies of the reduced form of these proteins suggest that the heme environments of the unmodified monomer and disulfide dimer derivatives of iso-1 ferrocytochrome c are similar and indicate that treatment of the thionitrobenzoate-derivatized and disulfide dimer forms of the protein with sodium dithionite results in cleavage of the disulfide bonds at position 102. Circular dichroism studies reveal that only the disulfide dimer form of iso-1 ferricytochrome c exhibits a Soret CD spectrum which differs from the native, unmodified monomer in that the intensity of the negative band at approximately 420 nm is diminished in the spectrum of the dimer relative to the spectrum of the monomer. Soret CD spectra of the ascorbate-reduced form of all protein derivatives are similar. The process of "autoreduction" of yeast iso-1 ferricytochrome c is shown to occur in the absence of a free sulfhydryl group at position 102 and is exacerbated under moderately high pH conditions. These results are suggestive of the presence of a redox-active amino acid, perhaps a tyrosine, in yeast iso-1 cytochrome c.

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Section: Discussionsupporting

confidence: 78%

Section: Temperature Dependence Of Selected Hyperfine-shifted Proton mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

Moench¹,

Satterlee²

1995

J Protein Chem

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“…These data, however, do not agree as well with other studies (Hickey et al, 1988;Das et al, 1989) that place the Tm of this protein near 46 OC. The lower T m may be due to the presence of dimerized protein (Bryant et al, 1985). WT protein, the (+) C102T variant, and the (0) V20C variant at pH 7.2 and 300 K. The curves represent the nonlinear least-squares fits of the data to the two-state hypothesis (Santoro & Bolen, 1988).…”

Section: Resultsmentioning

confidence: 99%

Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state

Betz

Pielak²

1992

Biochemistry

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We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c. The disulfide spontaneously forms upon purification. A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls. Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process. Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6. Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls. The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature. The cross-link also decreases the pH dependence of these observables. Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant. We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.

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“…a Swint and Robertson, 1993;' O'Neil et al, 1995;Hurle et al, 1990;Viguera et al, 1994; e Jackson et al, 1993;'Akke and Forsen, 1990;Khorasanizadeh et al, 1993;Wintrode et al, 1994;Scholtz, 1995;J Agashe and Udgaonkar, 1995;Lim et al, 1992; ' Marqusee and Sauer, 1994;McLendon and Smith, 1978;Hagihara et al, 1994; O Privalov and Gill, 1988;P Pace et al, 1990;Shirley et al, 1992;Yu et al, 1994; ' Bowie and Sauer, 1989; ' Egan et al, 1993;Ramdas et al, 1986;"Bryant et al, 1985; " Cohen and Pielak, 1994;x Kelley et al, 1987;Santor0 and Bolen, 1992;'Clarke and Fersht, 1993; aa Pace et al, 1992;" Griko et al, 1994; cc Greene and Pace, 1974;dd Munson et al, 1994;Filimonov et al, 1993; f f Saito and Wada, 1983; gg Ahmad and Bigelow, 1982; hh Taniyama et al, 1992; I' Herning et al, 1992;'' Ropson et al, 1990; kk Shortle and Meeker, 1986;"Carra et al, 1994; mm Craig et al, 1987; "" Makhatadze et al, 1994;O0 De Young et al, 1993...…”

Section: Aasamentioning

confidence: 99%

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

1995

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Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (AC,), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and AC,, Keywords: denaturation; guanidine hydrochloride; heat capacity changes; m values; protein folding; protein stability; solvent-accessible surface area; urea It has been known for many years that proteins can be unfolded in aqueous solution by high concentrations of certain reagents such as guanidine hydrochloride or urea. Denaturation with these chemicals is one of the primary ways of measuring the conformational stability of proteins and comparing the stabilities of mutant proteins. The use of these two denaturants is extremely widespread (Pace, 1986), even though the exact nature of the molecular interaction of denaturant molecules with protein surfaces is not well understood. It is known from solubility and transfer experiments with model compounds that the interaction of urea and Gdn HCI with the constituent groups of proteins is more favorable than the interaction of those groups with water (Tanford, 1970). These denaturants alter the equi-~"

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Refolding a disulfide dimer of cytochrome c

Cited by 20 publications

References 16 publications

A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

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