Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor-V

doi:10.1110/ps.26801

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Disulfide bond effects on protein stability: Designed variants of Cucurbita maxima trypsin inhibitor-V

Maria Zavodszky¹, Chao-Wei Chen², Jenq-Kuen Huang², Michal Zolkiewski¹, Lisa Wen² and Ramaswamy Krishnamoorthi¹

¹ Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA
² Department of Chemistry, Western Illinois University, Macomb, Illinois 61455, USA

Reprint request to: Ramaswamy Krishnamoorthi, Department of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, Kansas 66506, USA; e-mail: krish{at}ksu.edu; fax: 785-532-7278.

Attempts to increase protein stability by insertion of noveldisulfide bonds have not always been successful. According tothe two current models, cross-links enhance stability mainlythrough denatured state effects. We have investigated the effectsof removal and addition of disulfide cross-links, protein flexibilityin the vicinity of a cross-link, and disulfide loop size onthe stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V;7 kD) by differential scanning calorimetry. CMTI-V offers theadvantage of a large, flexible, and solvent-exposed loop notinvolved in extensive intra-molecular interactions. We haveuncovered a negative correlation between retention time in hydrophobiccolumn chromatography, a measure of protein hydrophobicity,and melting temperature (T_m), an indicator of native state stabilization,for CMTI-V and its variants. In conjunction with the completeset of thermodynamic parameters of denaturation, this has ledto the following deductions: (1) In the less stable, disulfide-removedC3S/C48S ( ${Delta}$ ${Delta}$ G_d^50°C = -4 kcal/mole; ${Delta}$ T_m = -22°C), the nativestate is destabilized more than the denatured state; this alsoapplies to the less-stable CMTI-V* ( ${Delta}$ ${Delta}$ G_d^50°C = -3 kcal/mole; ${Delta}$ T_m = -11°C), in which the disulfide-containing loop is openedby specific hydrolysis of the Lys⁴⁴-Asp⁴⁵ peptide bond; (2)In the less stable, disulfide-inserted E38C/W54C ( ${Delta}$ ${Delta}$ G_d^50°C= -1 kcal/mole; ${Delta}$ T_m = +2°C), the denatured state is morestabilized than the native state; and (3) In the more stable,disulfide-engineered V42C/R52C ( ${Delta}$ ${Delta}$ G_d^50°C = +1 kcal/mole; ${Delta}$ T_m= +17°C), the native state is more stabilized than the denaturedstate. These results show that a cross-link stabilizes bothnative and denatured states, and differential stabilizationof the two states causes either loss or gain in protein stability.Removal of hydrogen bonds in the same flexible region of CMTI-Vresulted in less destabilization despite larger changes in theenthalpy and entropy of denaturation. The effect of a cross-linkon the denatured state of CMTI-V was estimated directly by meansof a four-state thermodynamic cycle consisting of native anddenatured states of CMTI-V and CMTI-V*. Overall, the resultsshow that an enthalpy-entropy compensation accompanies disulfidebond effects and protein stabilization is profoundly modulatedby altered hydrophobicity of both native and denatured states,altered flexibility near the cross-link, and residual structurein the denatured state.

Keywords: Disulfide-bond; cross-link; protein stability; differential scanning calorimetry; denaturation; folding

Abbreviations: CMTI-V, Cucurbita maxima trypsin inhibitor-V • DSC, differential scanning calorimetry • ${Delta}$ C_p, heat capacity change • T_m, melting temperature • CD, circular dichroism • HPLC, high pressure liquid chromatography • SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis • DTNB, 5,5' -dithiobis (2-nitrobenzoic acid).

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