Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin‐4

• Published in: Protein science Vol. 15; no. 1; pp. 33 - 44
• Main Authors: Vaz, Daniela C., Rodrigues, J. Rui, Sebald, Walter, Dobson, Christopher M., Brito, Rui M.M.
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.01.2006
• Subjects: ANS, 8‐anilino‐1‐naphthalenesulfonate; C24T‐IL4, Interleukin‐4 with cysteine 24 replaced by threonine; C3T‐IL4, Inter‐leukin‐4 with cysteine 3 replaced by threonine; CD, circular dichroism; Circular Dichroism; conformational stability; disulfide bridges; Disulfides - chemistry; enthalpy; Entropy; four‐helix bundle; GdmCl, guanidinium chloride; GM‐CSF, granulocyte‐macrophage colony stimulating factor; G‐CSF, granulocyte colony stimulating factor; Humans; Hydrophobic and Hydrophilic Interactions; IL2, Interleukin‐2; IL4, Interleukin‐4; Interleukin-4 - chemistry; Interleukin-4 - genetics; Interleukin‐4; Magnetic Resonance Spectroscopy; NMR, nuclear magnetic resonance; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; SASA, solvent‐accessible surface area; Spectrometry, Fluorescence; Surface Properties; Temperature; thermal unfolding; Tm, transition temperature of unfolding; TS, temperature of maximal stability; Urea; WT‐IL4, wild type Interleukin‐4; ΔCp, change in heat capacity upon protein unfolding; ΔG(H2O), conformational stability; ΔHm, enthalpy change of unfolding at the transition temperature; ΔSm, entropy change of unfolding at the transition temperature
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.051593306

The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin‐4 (IL4), a four‐helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T‐IL4 and C24T‐IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent‐accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild‐type protein. Transition temperatures of unfolding decreased 14°C for C3T‐IL4 and 10°C for C24T‐IL4, when compared to WT‐IL4, and the conformational stability, at 25°C, decreased 4.9 kcal/mol for C3T‐IL4 and 3.2 kcal/mol for C24T‐IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle.