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

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 novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column 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 complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (G_d^50°C = -4 kcal/mole; T_m = -22°C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (G_d^50°C = -3 kcal/mole; T_m = -11°C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys⁴⁴-Asp⁴⁵ peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (G_d^50°C = -1 kcal/mole; T_m = +2°C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (G_d^50°C = +1 kcal/mole; T_m = +17°C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in 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 • 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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