Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates.


Abstract

The equilibrium denaturation of an Escherichia coli-derived recombinant murine interleukin-6 (mIL-6) was studied using fluorescence and circular dichroism spectroscopy. The urea-induced unfolding of mIL-6 at pH 4.0 can be described by a two-state unfolding mechanism based on the superimposibility of the CD and fluorescence unfolding transitions. Assuming a two-state mechanism and a linear dependence of the free energy of unfolding on denaturant concentration, a value of 6.9-9.0 kcal/mol was calculated for the free energy of unfolding in the absence of denaturant [delta GU(H2O)]. However, when GuHCl was used as a denaturant at pH 4.0, a biphasic unfolding transition was observed. This unfolding transition has a distinct midpoint occurring at 2.5 M GuHCl, which is indicative of the formation of stable folding intermediates. Similar intermediate folded species were also observed at pH 7.4 when either urea or GuHCl were used as denaturants. The intermediate folded states of mIL-6 exhibited a tendency to aggregate, as judged by the concentration dependence of their fluorescence characteristics. The fluorescence emission maximum of mIL-6 at pH 7.4 in the presence of 1.5 M GuHCl, for example, was blue-shifted from 343 nm at a protein concentration of 50 micrograms/mL to 336 nm at 500 micrograms/mL. Intermediate formation at pH 4.0, using 10 mM sodium acetate buffer and urea as the denaturant, was facilitated by the addition of 0.4 and 0.8 M salt, where the salt was either NaCl or GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4929, 4930, 4931, 4932 Extra Details: two-state unfolding mechanism; biphasic unfolding transition;,concentration dependence; folding intermediates

Submission Details

ID: vozZLc5P3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Ward LD;Matthews JM;Zhang JG;Simpson RJ,Biochemistry (1995) Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates. PMID:7547897
Additional Information

Structure view and single mutant data analysis

Study data

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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
2L3Y 2011-09-28 Solution structure of mouse IL-6

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Interleukin-6 P08505 IL6_MOUSE