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Publication
Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of Interleukin-4
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Advisor(s)
Abstract(s)
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 disulfidesin 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 solventaccessible 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 14C
for C3T-IL4 and 10C for C24T-IL4, when compared toWT-IL4, and the conformational stability, at 25C,
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.
Description
Keywords
Interleukin-4 Four-helix bundle Conformational stability Disulfide bridges Urea Thermal unfolding Enthalpy Entropy