Analysis of the conformation and stability of Escherichia coli derived recombinant human interleukin 4 by circular dichroism.


Abstract

The conformation and stability of Escherichia coli derived recombinant human interleukin 4 (rhuIL-4) have been examined by circular dichroism (CD). Protein unfolding was detected by ellipticity changes at 222 nm with increasing concentrations of guanidine hydrochloride (GdnHCl). The unfolding midpoint ([GdnHCl]1/2) was 3.7 M, the free energy of unfolding, (delta GDH2O), was 5.9 kcal/mol and the dependence of delta GD on the GdnHCl concentration (m) was 1.6 (kcal/mol)/M. This unfolding was demonstrated to be reversible upon removal of the GdnHCl by dialysis. Analysis of the far-UV CD spectrum indicated the presence of a high percentage of alpha-helical structure (ca. 73%). A small change in ellipticity was noted over the pH range 1.9-9.6, suggesting that the protein undergoes a minor conformational change with an apparent pKa of 4.17. Virtually complete biological activity, measured in vitro in a T-cell proliferation assay, was recovered following exposure to extreme values of pH (i.e., pH 3 and 10). An analysis of the near-UV CD spectrum indicated that the single tryptophan residue at position 91 was unconstrained and most likely exposed to the solvent. Titration with 4,4'-dithiodipyridine and 2-nitrothiosulfobenzoate established that the six cysteine residues in rhuIL-4 were involved in intramolecular disulfide linkages. These data support that rhuIL-4 has a highly stable three-dimensional structure. Study holds ProTherm entries: 4245 Extra Details: dialysis; alpha-helical structure; conformational change;,biological activity; disulfide linkages

Submission Details

ID: m2dWEJdb

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Windsor WT;Syto R;Le HV;Trotta PP,Biochemistry (1991) Analysis of the conformation and stability of Escherichia coli derived recombinant human interleukin 4 by circular dichroism. PMID:1991106
Additional Information

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
1ITI 1993-07-15 THE HIGH RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 DETERMINED BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
2CYK 1994-12-20 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
1BBN 1993-10-31 THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 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
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
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
2B8U 2006-05-30 1.8 Crystal structure of wildtype human Interleukin-4
2B8Y 2006-05-30 1.8 Crystal structure of the interleukin-4 variant T13DF82D
2B91 2006-05-30 2.0 Crystal structure of the interleukin-4 variant F82DR85A
4YDY 2015-03-04 2.0 CRYSTAL STRUCTURE OF DARPIN 44C12V5 IN COMPLEX WITH HUMAN IL-4
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
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
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
97.4 Interleukin-4 P79339 IL4_MACFA
100.0 Interleukin-4 Q8HYB1 IL4_PANTR
100.0 Interleukin-4 P05112 IL4_HUMAN