Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates.
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)
Ward
L D
LD
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
Matthews
J M
JM
Zhang
J G
JG
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D015850:Interleukin-6; D011994:Recombinant Proteins; D012965:Sodium Chloride; D019791:Guanidine