Fluorescence labeling-based differential scanning fluorimetry, an effective method for protein thermal stability and protein-compound binding analysis.

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and protein-ligand interaction. However, its utility is often limited by the presence of detergents, which can affect hydrophobic binding. To tackle this issue, we developed an effective fluorescence-labeled DSF (FL-DSF) technique that tracks protein denaturation by monitoring the labeling fluorescence decrease, thus overcoming challenges typically encountered with traditional DSF methods. In this research, FL-DSF was first validated using Peroxisome Proliferators-Activated Receptor γ (PPARγ), Retinoid X Receptor α (RXRα), and Lysozyme, confirming its accuracy in determining melting curves. Expectedly, FL-DSF also exhibited strong compatibility with detergents in our investigations. Besides this, a new calculation method was proposed to characterize the protein denaturation process and evaluate protein-ligand binding. This mathematical model goes beyond traditional approaches, which simply treated the melting temperature (T) shift as a concentration-dependent variable. Instead, it comprehensively incorporates the influence of irreversible denaturation-induced native protein loss on the equilibrium of protein-ligand binding. This methodology was successfully applied into the evaluation of binding affinity for 2 classical binding systems of PPARγ-Rosiglitazone and RXRα-CD3254. It was also utilized for the following binding screening studies, leading to the discovery of promising ligands for PPARγ, RXRα, and Lysozyme.