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

Abstract

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 highresolution 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 solventexposed 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.

Trastuzumab-Peptide Interactions: Mechanism and Application in Structure-Based Ligand Design

“…This indicates that binding of the protein leads to an increase in the conformation stability within these interaction regions. These predictions agree with the experimental findings that the interaction of proteins with ligands often takes place with an increase in protein stability and structural order [31,32]. That is to say, formation of the Fab-octadecapeptide complex makes the binding site, namely, loop regions of the octadecapeptide and the binding groove of Fab in this system more stable than other parts.…”

High throughput thermostability screening of monoclonal antibody formulations

“…Currently the interaction of SYPRO Orange with specific proteins is not thoroughly understood, due primarily to the fact that the supplier does not provide information on the chemical structure or molecular mass of the probe. It is possible that the interaction of SYPRO Orange can be driven not only by hydrophobicity of the protein but also by charges on the protein, as is sometimes the case for 8‐anilino‐1‐naphthalene sulfonate (ANS), another fluorescent probe sensitive to hydrophobicity 16, 28. Unfortunately ANS could not be assessed in the DSF format due to the absence of any appropriate filters for the collection of ANS fluorescence.…”

pH‐dependent Interaction of Rhodopsin with Cyanidin‐3‐glucoside. 1. Structural Aspects†

“…We obtained values on the order of 50–100 μ m indicating moderate to weak binding of C3G to dark‐state rhodopsin at pH 6. The affinity is comparable to, for example, ANS binding to proteins (33), a ligand that binds with relatively broad specificity to molten globule‐like states of proteins. Thus, the anthocyanin ligand investigated here is certainly not a high‐affinity ligand that can compare in affinity to rhodopsin’s retinal ligands.…”