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.

Submission Details

ID: FbDXM33D

Submitter: Shu-Ching Ou

Submission Date: Dec. 6, 2018, 6:39 p.m.

Version: 1

Publication Details
Vaz DC;Rodrigues JR;Sebald W;Dobson CM;Brito RM,Protein Sci (2006) Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of interleukin-4. PMID:16373475
Additional Information

Study Summary

Number of data points 150
Proteins Interleukin-4
Unique complexes 3
Assays/Quantities/Protocols Experimental Assay: Binding: kon ; Experimental Assay: Binding: koff ; Experimental Assay: Stability: ΔG at 0.5 °C ; Experimental Assay: Stability: ΔG at 5 °C ; Experimental Assay: Stability: ΔG at 10 °C ; Experimental Assay: Stability: ΔG at 15 °C ; Experimental Assay: Stability: ΔG at 20 °C ; Experimental Assay: Stability: ΔG at 25 °C ; Experimental Assay: Stability: ΔG at 30 °C ; Experimental Assay: Stability: ΔG at 35 °C ; Experimental Assay: Stability: ΔG at 40 °C ; Experimental Assay: Stability: m at 0.5 °C ; Experimental Assay: Stability: m at 5 °C ; Experimental Assay: Stability: m at 10 °C ; Experimental Assay: Stability: m at 15 °C ; Experimental Assay: Stability: m at 20 °C ; Experimental Assay: Stability: m at 25 °C ; Experimental Assay: Stability: m at 30 °C ; Experimental Assay: Stability: m at 35 °C ; Experimental Assay: Stability: m at 40 °C ; Experimental Assay: Stability: Tm ; Experimental Assay: Stability: ΔG at 37 °C ; Derived Quantity: SD of Binding: kon ; Derived Quantity: SD of Binding: koff ; Derived Quantity: Binding: Kd ; Derived Quantity: SD of Stability: ΔG at 0.5 °C ; Derived Quantity: SD of Stability: ΔG at 5 °C ; Derived Quantity: SD of Stability: ΔG at 10 °C ; Derived Quantity: SD of Stability: ΔG at 15 °C ; Derived Quantity: SD of Stability: ΔG at 20 °C ; Derived Quantity: SD of Stability: ΔG at 25 °C ; Derived Quantity: SD of Stability: ΔG at 30 °C ; Derived Quantity: SD of Stability: ΔG at 35 °C ; Derived Quantity: SD of Stability: ΔG at 40 °C ; Derived Quantity: SD of Stability: m at 0.5 °C ; Derived Quantity: SD of Stability: m at 5 °C ; Derived Quantity: SD of Stability: m at 10 °C ; Derived Quantity: SD of Stability: m at 15 °C ; Derived Quantity: SD of Stability: m at 20 °C ; Derived Quantity: SD of Stability: m at 30 °C ; Derived Quantity: SD of Stability: m at 35 °C ; Derived Quantity: SD of Stability: m at 40 °C ; Derived Quantity: SD of Stability: m at 25 °C ; Derived Quantity: Stability: Cm at 0.5 °C ; Derived Quantity: SD of Stability: Cm at 0.5 °C ; Derived Quantity: Stability: Cm at 5 °C ; Derived Quantity: SD of Stability: Cm at 5 °C ; Derived Quantity: SD of Stability: Cm at 10 °C ; Derived Quantity: Stability: Cm at 10 °C ; Derived Quantity: Stability: Cm at 15 °C ; Derived Quantity: SD of Stability: Cm at 15 °C ; Derived Quantity: Stability: Cm at 20 °C ; Derived Quantity: SD of Stability: Cm at 20 °C ; Derived Quantity: Stability: Cm at 25 °C ; Derived Quantity: SD of Stability: Cm at 25 °C ; Derived Quantity: Stability: Cm at 30 °C ; Derived Quantity: SD of Stability: Cm at 30 °C ; Derived Quantity: Stability: Cm at 35 °C ; Derived Quantity: SD of Stability: Cm at 35 °C ; Derived Quantity: SD of Stability: Cm at 40 °C ; Derived Quantity: Stability: Cm at 40 °C ; Derived Quantity: Stability: Ts ; Derived Quantity: Stability: ΔSm ; Derived Quantity: Stability: ΔG at the temperature of maximal stability ; Computational Protocol: Stability: ΔHm ; Computational Protocol: Stability: ΔCp
Libraries Variants for Interleukin-4_Receptor-Alpha-Chain

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1IAR 1999-02-25T00:00:00+0000 2.3 INTERLEUKIN-4 / RECEPTOR ALPHA CHAIN COMPLEX
1IRS 1996-03-22T00:00:00+0000 0 IRS-1 PTB DOMAIN COMPLEXED WITH A IL-4 RECEPTOR PHOSPHOPEPTIDE, NMR, MINIMIZED AVERAGE STRUCTURE
3BPL 2007-12-18T00:00:00+0000 2.93 Crystal structure of the IL4-IL4R-Common Gamma ternary complex
3BPN 2007-12-18T00:00:00+0000 3.02 Crystal structure of the IL4-IL4R-IL13Ra ternary complex
3BPO 2007-12-18T00:00:00+0000 3.0 Crystal structure of the IL13-IL4R-IL13Ra ternary complex
5E4E 2015-10-05T00:00:00+0000 3.0 Engineered Interleukin-13 bound to receptor
6OEL 2019-03-27T00:00:00+0000 3.1 Engineered Fab bound to IL-4 receptor
6WGL 2020-04-05T00:00:00+0000 2.82 Dupilumab fab with Crystal Kappa design complexed with human IL-4 receptor
1BBN 1992-05-01T00:00:00+0000 0 THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1BCN 1992-05-01T00:00:00+0000 0 THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 B Interleukin-4 P24394 IL4RA_HUMAN
96.9 A Interleukin-4 P79339 IL4_MACFA
100.0 A Interleukin-4 Q8HYB1 IL4_PANTR
100.0 A Interleukin-4 P05112 IL4_HUMAN