Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32.

D R Hickey,G Mclendon,F Sherman

doi:10.1016/s0021-9258(19)81359-5

D R Hickey, G Mclendon + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(19)81359-5

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Abstract

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.

Highlights

Summary-Iso-1-cytochromes c having Leu-32, Gln-32, Tyr-32, and Trp-32 replacements for Lys-32 were extracted from strains of S. cereuisiae, chemically modified at Cys-107, and guanidine and thermally unfolded in multistateequilibria
The unfolding free energy changes for each protein depend on the denaturation method, the relative orders of stability do not: Lys-32-SCH3= Leu-32-SCH3>Gln-32-SCH3 > Tyr-32-SCH3 = Trp-32-SCH3
Replacement of Lys-32 resulted in a range of stabilities suggesting that small structural changes may significantly affect protein stability

Summary

Thermodynamic Stabilitiesof Altered Proteins

E, normalized to the absorbance at 410 nm as a function of Melting curves were analyzed as follows:upper and lower base-line wavelength are shown for all fiveblockedoxidized iso-1 slopes and intercepts were first determinedwith a linear least program. 32-SCH3, Leu-32-SCH3o, r Tyr-32-SCH3.The 695 nm bands where A(T )is the measured absorbance at temperature T,and AN(T ) (Fig. 2B) have apparent molar extinction coefficients whose and AD(T )are calculated absorbances at temperature T for the native values (beforebackground subtraction) follow the order: Lysand denatured states, respectively. Trp-32-SCH3.This band originates from ligation of the heme iron (Schechter and Saludjian, 1967) and has been used to assess the conformational integrity of the heme crevice by subtraction of the background absorbance at thawt avelength (Kaminsky et al, 1973) If this background subtraction is the equation (Becktel and Schellman, 1987): performed for the iso-l-SCH3 proteins, their 695 nm bands. Iso-1, respectively, and AT is the T , difference for bothproteins (Becktel and Schellman, 1987).No corrections to the datwa ere made for volume expansion

RESULTS

Thermodynamic Stabilities of Altered Proteins i t

DISCUSSION

Comparison of relative growth rates and thermal stabilities

In vitro