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

Attempts to increase protein stability by insertion of novel disulfide bond s have not always been successful. According to the two current models, cro ss-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 siz e on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) b y differential scanning calorimetry. CMTI-V offers the advantage of a large , flexible, and solvent-exposed loop not involved in extensive intra-molecu lar interactions. We have uncovered a negative correlation between retentio n time in hydrophobic column chromatography, a measure of protein hydrophob icity, and melting temperature (T-m), an indicator of native state stabiliz ation, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following de ductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta DeltaG( d)(50 degreesC) = -4 kcal/mole; DeltaT(m) = -22 degreesC), the native state is destabilized more than the denatured state; this also applies to the le ss-stable CMTI-V* (Delta DeltaG(d)(50 degreesC) = -3 kcal/mole; DeltaT(m) = -11 degreesC), in which the disulfide-containing loop is opened by specifi c hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, d isulfide-inserted E38C/W54C (Delta DeltaG(d)(50 degreesC) = -1 kcal/mole; D eltaT(m) = +2 degreesC), the denatured state is more stabilized than the na tive state; and (3) In the more stable, disulfide-engineered V42C/R52C (Del ta DeltaG(d)(50 degreesC) = +1 kcal/mole; DeltaT(m) = +17 degreesC), the na tive state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differen tial 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 cy cle consisting of native and denatured states of CMTI-V and CMTI-V*. Overal l, the results show that an enthalpy-entropy compensation accompanies disul fide bond effects and protein stabilization is profoundly modulated by alte red hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.