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

¹ Centro de Neurociências de Coimbra, Universidade de Coimbra, 3004-517 Coimbra, Portugal
² Theodor-Boveri-Institut fur Biowissenschaften, Biozentrum, Physiologische Chemie II, Universitat Würzburg, D-97074 Würzburg, Germany
³ Department of Chemistry, Cambridge University, Cambridge CB2 1EW, United Kingdom
⁴ Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal

(RECEIVED May 17, 2005; FINAL REVISION October 4, 2005; ACCEPTED October 12, 2005)

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.

Keywords: Interleukin-4; four-helix bundle; conformational stability; disulfide bridges; urea; thermal unfolding; enthalpy; entropy

Abbreviations: 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 • G-CSF, granulocyte colony stimulating factor • GdmCl, guanidinium chloride • GM-CSF, granulocyte-macrophage colony stimulating factor • IL2, Interleukin-2 • IL4, Interleukin-4 • NMR, nuclear magnetic resonance • WT-IL4, wild type Interleukin-4 • C_p, change in heat capacity upon protein unfolding • G(H₂O), conformational stability • H_m, enthalpy change of unfolding at the transition temperature • S_m, entropy change of unfolding at the transition temperature • SASA, solvent-accessible surface area • T_m, transition temperature of unfolding • T_S, temperature of maximal stability

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051593306.

Reprint requests to: Rui M.M. Brito, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal; e-mail: brito{at}ci.uc.pt; fax: +351-239-827703.

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