Size-Dependent Relationships between Protein Stability and Thermal Unfolding Temperature Have Important Implications for Analysis of Protein Energetics and High-Throughput Assays of Protein–Ligand Interactions

Changes in protein stability are commonly reported as changes in the melting temperature, ΔT_M, or as changes in unfolding free energy at a particular temperature, ΔΔG°. Using data for 866 mutants from 16 proteins, we examine the relationship between ΔΔG° and ΔT_M. A linear relationship is observed for each protein. The slopes of the plots of ΔT_M vs ΔΔG° for different proteins scale as N^–1, where N is the number of residues in the protein. Thus, a given change in ΔG° causes a much larger change in T_M for a small protein relative to the effect observed for a large protein. The analysis suggests that reasonable estimates of ΔΔG° for a mutant can be obtained by interpolating measured values of T_M. The relationship between ΔΔG° and ΔT_M has implications for the design and interpretation of high-throughput assays of protein–ligand binding. So-called thermal shift assays rely upon the increase in stability which results from ligand binding to the folded state. Quantitative relationships are derived which show that the observed thermal shift, ΔT_M, scales as N^–1. Hence, thermal shift assays are considerably less sensitive for ligand binding to larger proteins.