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

Abstract

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 degrees C for C3T-IL4 and 10 degrees C for C24T-IL4, when compared to WT-IL4, and the conformational stability, at 25 degrees 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.

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

“…As expected, this value is significantly smaller than the Gibbs free energy of global protein unfolding which is reported to be in the range 18-40 kJ mol 21 (Refs. 3,4,32,33). On the other hand, the value of DG 0 derived for BSA is about 2.6 times lower than that reported for the two least reactive S-S bridges in BPTI 31 thus suggesting that the disulfide bridges in BSA are on average more exposed to the solvent.…”

Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14

“…The presence of a single disulfide bridge between residue Cys 3 and Cys 14 in somatostatin-14 makes it a simple yet attractive model system to probe altered conformational dynamics and aggregation in response to slight modifications in native structure (S-S bond cleavage). Disulfide bonds have been previously reported to play a key role in modulating the intrinsic dynamics of proteins that are crucial to their native function (10,67,68). In somatostatin and its peptidic analogs, the importance of the disulfide bond on its structural and aggregation features has been demonstrated previously (69,70).…”

“…6). Disulfide bond has previously been suggested to reduce conformational dynamics, increase mechanical stability, and reduce entropy in proteins/peptides (10,67,71). Additionally, this linkage has been shown to play a role in minimizing protein aggregation (14,15,72) thereby making it an effective modulator of aggregation.…”

Getting specificity from simplicity in putative proteins from the prebiotic Earth

“…These stability values are modest; they are slightly higher than those determined for the lac repressor DNA binding domain (7) but less than the corresponding values for phage 434 Cro protein (8) and most other small, all-helical proteins. The conformational stability of CG 3 KIA7 is significantly higher, Ϸ5 kcal/mol, and similar to variants of IL-4, which also contain two disulfide bonds (9). It is remarkable that the stabilizing interactions in KIA7 are able to pay the substantial entropic penalty of oligomerization (4.5 kcal/mol) (10) and still be as stable as some single-chain proteins, which do not pay this penalty.…”