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
1IRS 1997-05-15 IRS-1 PTB DOMAIN COMPLEXED WITH A IL-4 RECEPTOR PHOSPHOPEPTIDE, NMR, MINIMIZED AVERAGE STRUCTURE
1BBN 1993-10-31 THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1ITI 1993-07-15 THE HIGH RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 DETERMINED BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1CYL 1994-09-30 ASPECTS OF RECEPTOR BINDING AND SIGNALLING OF INTERLEUKIN-4 INVESTIGATED BY SITE-DIRECTED MUTAGENESIS AND NMR SPECTROSCOPY
1BCN 1993-10-31 THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1ITM 1994-05-31 ANALYSIS OF THE SOLUTION STRUCTURE OF HUMAN INTERLEUKIN 4 DETERMINED BY HETERONUCLEAR THREE-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE TECHNIQUES
1ITL 1993-04-15 HUMAN INTERLEUKIN 4: THE SOLUTION STRUCTURE OF A FOUR-HELIX-BUNDLE PROTEIN
2CYK 1994-12-20 ASPECTS OF RECEPTOR BINDING AND SIGNALLING OF INTERLEUKIN-4 INVESTIGATED BY SITE-DIRECTED MUTAGENESIS AND NMR SPECTROSCOPY
2D48 2006-05-30 1.65 Crystal structure of the Interleukin-4 variant T13D
2B8X 2006-05-30 1.7 Crystal structure of the interleukin-4 variant F82D
2B8Y 2006-05-30 1.8 Crystal structure of the interleukin-4 variant T13DF82D
2B8U 2006-05-30 1.8 Crystal structure of wildtype human Interleukin-4
4YDY 2015-03-04 2.0 CRYSTAL STRUCTURE OF DARPIN 44C12V5 IN COMPLEX WITH HUMAN IL-4
2B91 2006-05-30 2.0 Crystal structure of the interleukin-4 variant F82DR85A
1HZI 2001-08-29 2.05 INTERLEUKIN-4 MUTANT E9A
2B90 2006-05-30 2.1 Crystal structure of the interleukin-4 variant T13DR85A
1RCB 1993-10-31 2.25 CRYSTAL STRUCTURE OF HUMAN RECOMBINANT INTERLEUKIN-4 AT 2.25 ANGSTROMS RESOLUTION
1IAR 2000-03-03 2.3 INTERLEUKIN-4 / RECEPTOR ALPHA CHAIN COMPLEX
2INT 1994-01-31 2.35 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN INTERLEUKIN-4
2B8Z 2006-05-30 2.5 Crystal structure of the interleukin-4 variant R85A
5FHX 2016-03-30 2.55 CRYSTAL STRUCTURE OF CODV IN COMPLEX WITH IL4 AT 2.55 Ang. RESOLUTION.
1HIK 1996-01-29 2.6 INTERLEUKIN-4 (WILD-TYPE)
3BPL 2008-02-05 2.93 Crystal structure of the IL4-IL4R-Common Gamma ternary complex
5E4E 2015-12-02 3.0 Engineered Interleukin-13 bound to receptor
3BPO 2008-02-05 3.0 Crystal structure of the IL13-IL4R-IL13Ra ternary complex
1HIJ 1996-01-29 3.0 INTERLEUKIN-4 MUTANT WITH ARG 88 REPLACED WITH GLN (R88Q)
3BPN 2008-02-05 3.02 Crystal structure of the IL4-IL4R-IL13Ra ternary complex
6OEL 2019-08-07 3.1 Engineered Fab bound to IL-4 receptor
3QB7 2012-04-25 3.24 Interleukin-4 mutant RGA bound to cytokine receptor common gamma

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