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

• Published in: The journal of physical chemistry. B Vol. 122; no. 21; pp. 5278 - 5285
• Main Authors: Watson, Matthew D, Monroe, Jeremy, Raleigh, Daniel P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 31-05-2018
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.7b05684

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, ΔΔ G°. Using data for 866 mutants from 16 proteins, we examine the relationship between ΔΔ G° and ΔT M. A linear relationship is observed for each protein. The slopes of the plots of ΔT M 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 T M 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 T M. The relationship between ΔΔ G° 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.