Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist.
Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25 degrees C and 37 degrees C. Overall, the affinity of ANS was lower at 37 degrees C compared to 25 degrees C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.
Latypov
Ramil F
RF
Department of Pharmaceutics, Amgen Inc., Seattle, Washington 98119-3105, USA. rlatypov@amgen.com
Liu
Dingjiang
D
Gunasekaran
Kannan
K
Harvey
Timothy S
TS
Razinkov
Vladimir I
VI
Raibekas
Andrei A
AA
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D053590:Interleukin 1 Receptor Antagonist Protein; D014508:Urea