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Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

¹ Department of Pharmaceutics, Amgen Inc., Seattle, Washington 98119-3105, USA
² Department of Pharmaceutics, Amgen Inc., Thousand Oaks, California 91320-1799, USA
³ Department of Protein Science, Amgen Inc., Seattle, Washington 98119-3105, USA
⁴ Department of Protein Science, Amgen Inc., Thousand Oaks, California 91320-1799, USA
⁵ Department of Global Cellular and Analytical Resources, Amgen Inc., Seattle, Washington 98119-3105, USA

(RECEIVED November 2, 2007; FINAL REVISION December 18, 2007; ACCEPTED December 19, 2007)

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°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°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.

Keywords: protein–ligand interactions; protein stability; protein aggregation; NMR

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