Evidence for an acid-induced molten-globule state in interleukin-2; a fluorescence and circular dichroism study.
The effect of low pH on the secondary and tertiary structure of the monomeric single-disulphide protein interleukin-2 (IL2) was monitored by fluorescence and circular dichroism spectroscopy. Between pH 4 and pH 2 there is a gradual loosening of the tertiary structure as revealed by changes in tyrosine and tryptophan fluorescence emission, tryptophan fluorescence anisotropy, accessibility to the fluorescence quencher acrylamide and aromatic circular dichroism. The overall molecular size and secondary structure content are not significantly changed by acidification. These data are consistent with a 'molten globule' state for IL2 at low pH, in which the hydrophobic core/secondary structure is largely intact but the tertiary structure is flexible. Similar effects to low pH are seen at sub-denaturing concentrations of guanidine hydrochloride. Analysis of fluorescence lifetimes and derivative emission spectra of the single tryptophan, Trp-121, shows the existence of two distinct orientations for this side-chain, one of which is affected by a quenching group (the effect of which diminishes upon acidification) and another which is essentially unquenched. The identity of the quenching group is unclear but may well be Cys-125. The formation of the molten globule titrates with a pKa of about 2.3; this is unusually low for the acidic groups in proteins and indicates a perturbed pKa of a residue involved in a structurally important interaction such as a salt bridge. Candidate residues are Glu-15 or Asp-20, close to His-16 on the N-terminal helix of IL-2.
Dryden
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Department of Biochemistry, University of Newcastle upon Tyne, U.K.
Weir
M P
MP
D000143:Acids; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D007376:Interleukin-2 / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D000143:Acids; D007376:Interleukin-2; D014443:Tyrosine; D014364:Tryptophan