Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment

Abstract

Protein‐ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein‐ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein‐ligand interactions in crowded cellular environments. In this study, we investigated the feasibility of induced circular dichroism (ICD) using 8‐anilinonaphthalene‐1‐sulfonic acid (ANS) for local conformational analysis at the binding site in crowded environments. Bovine serum albumin (BSA) concentration‐dependent measurements were performed to assess the feasibility of ANS‐ICD for analyzing protein interior binding sites. The results showed distinct changes in the ANS‐ICD spectra of BSA solutions, indicating their potential for analyzing the internal conformation of proteins. Moreover, temperature‐dependent measurements were performed in dilute and crowded environments, revealing distinct denaturation pathways of BSA binding sites. Principal component analysis of ANS‐ICD spectral changes revealed lower temperature pre‐denaturation in the crowded solution than that in the diluted solution, suggesting destabilization of binding sites owing to self‐crowding repulsive interactions. The established ANS‐ICD method can provide valuable conformational insights into protein‐ligand interactions in crowded cellular environments.