Stability of yeast iso‐1‐ferricytochrome c as a function of pH and temperature

Abstract

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-lferricytochrome c were performed between pH 3 and 5 . Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (AGD), enthalpy ( A H D ) , and entropy (AS,) of denaturation were determined as functions of pH and temperature. The value of AGD at 300 K, pH 4.6, is 5.1 k 0.3 kcal mol". The change in molar heat capacity upon denaturation (AC,), determined by the temperature dependence of AH, as a function of pH (1.37 k 0.06 kcal mol" K-'), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a AH, of 1.30 kcal mol" residue" at 373.6 K and a AS, of 4.24 cal mol" K" residue-' at 385.2 K. These values agree with those for other single-domain, globular proteins.Keywords: cytochrome c; equilibrium thermodynamics; protein denaturation; protein stability A goal of protein chemistry is to understand how interactions between amino acid residues control stability, structure, and function. Anfinsen and coworkers showed that the amino acid sequence of a protein determines its higher-order structure and Reprint requests to: Gary J. Pielak, Department of Chemistry, Campus Box 3290, University of North Carolina, Chapel Hill, North Carolina 27599; e-mail: gjpielakQuncvxl .oit.unc.edu.Abbreviations: Ah, T , absorbance at wavelength X (nm) and temperature T(K); bD, absorbance of the denatured-state baseline linearly extrapolated to 0 K; b N , absorbance of the native-state baseline linearly extrapolated to 0 K; C102T, the variant of yeast iso-1-cytochrome c in which the cysteine at position 102 in the wild-type protein is replaced by a threonine. The numbering system of higher eukaryotic cytochromes c is used (Cutler et al., 1987;Moore & Pettigrew, 1990); D, the denatured state; KO, T , the apparent equilibrium constant for denaturation at temperature T; mD, the slope of the D baseline; mN, the slope of the native-state baseline; N , the native state; Tm, the temperature at which half the protein molecules are denatured; yD, T , the absorbance due to the D baseline at temperature T; yN, T , the absorbance due to the N baseline at temperature T; CY, , T , the apparent fraction of D at temperature T; AC,, the heat capacity of D minus N, AG,, the apparent Gibbs free energy of denaturation; AGQT, AGD at temperature T; AH,,,, the calorimetric enthalpy of denaturation; A H D , the apparent (i.e., van't Hoff) enthalpy of denaturation; AHD, AHD at temperature C AH,,,, AHD,T,,,; ASD, the apparent entropy of denaturation; A S , T , AS, at temperature T; AS,,,, A S , Tm (equal to AH,,,/Tm); Av, the number of protons bound to D minus the number of protons bound to N, a,, the e...

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

“…AtCESA1CatD only (white circles) and in presence of UDP (black circles) and UDP-Glc (black squares) monitored at 222 nm. Solid lines are fit to a two-state model for thermal denaturation (Cohen and Pielak, 1994).…”

“…The thermal denaturation profiles were recorded at 222 nm from 25°C to 80°C at a heating rate of 1°C /min. The data were analyzed using a two-state model to determine the T m value (Cohen and Pielak, 1994).…”

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

“…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...…”

Thermal denaturation of iso-1-cytochromecvariants: Comparison with solvent denaturation

“…Multiple experiments were performed at each pH, allowing determination of the error in the measurement, since error from multiple experiments has been shown to significantly exceed error calculated directly from the non-linear least-squares fitting procedure (Cohen & Pielak, 1994). The means and standard deviations (or ranges) were calculated for AH,,, and T, at each pH and these values are shown for WT and variant proteins in Table 1.…”

“…Calculations were carried out at a temperature of 298 K and a pH of 4.5 to allow ready comparison with other thermal denaturation data for iso-1 -cytochrome c (Cohen & Pielak, 1994). The results are presented in Table 2.…”