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 1993-04-12T00:00:00+0000 0 THE HIGH RESOLUTION THREE-DIMENSIONAL SOLUTION STRUCTURE OF HUMAN INTERLEUKIN-4 DETERMINED BY MULTI-DIMENSIONAL HETERONUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1RCB 1992-08-26T00:00:00+0000 2.25 CRYSTAL STRUCTURE OF HUMAN RECOMBINANT INTERLEUKIN-4 AT 2.25 ANGSTROMS RESOLUTION
1ITM 1994-02-28T00:00:00+0000 0 ANALYSIS OF THE SOLUTION STRUCTURE OF HUMAN INTERLEUKIN 4 DETERMINED BY HETERONUCLEAR THREE-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE TECHNIQUES
2D48 2005-10-11T00:00:00+0000 1.65 Crystal structure of the Interleukin-4 variant T13D
4YDY 2015-02-23T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF DARPIN 44C12V5 IN COMPLEX WITH HUMAN IL-4
1ITL 1992-02-08T00:00:00+0000 0 HUMAN INTERLEUKIN 4: THE SOLUTION STRUCTURE OF A FOUR-HELIX-BUNDLE PROTEIN
1IAR 1999-02-25T00:00:00+0000 2.3 INTERLEUKIN-4 / RECEPTOR ALPHA CHAIN COMPLEX
3QB7 2011-01-12T00:00:00+0000 3.25 Interleukin-4 mutant RGA bound to cytokine receptor common gamma
2B8U 2005-10-10T00:00:00+0000 1.8 Crystal structure of wildtype human Interleukin-4
2INT 1993-07-22T00:00:00+0000 2.35 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN INTERLEUKIN-4

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