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, Δ TM , or as changes in unfolding free energy at a particular temperature, ΔΔ G°. Using data for 866 mutants from 16 proteins, we examine the relationship between ΔΔ G° and Δ TM . A linear relationship is observed for each protein. The slopes of the plots of Δ TM vs ΔΔ G° for different proteins scale as N-1 , where N is the number of residues in the protein. Thus, a given change in Δ G° causes a much larger change in TM for a small protein relative to the effect observed for a large protein. The analysis suggests that reasonable estimates of ΔΔ G° for a mutant can be obtained by interpolating measured values of TM . The relationship between ΔΔ G° and Δ TM 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, Δ TM , scales as N-1 . Hence, thermal shift assays are considerably less sensitive for ligand binding to larger proteins.