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

J Phys Chem B. 2018 May 31;122(21):5278-5285. doi: 10.1021/acs.jpcb.7b05684. Epub 2017 Sep 11.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Databases, Factual
  • Ligands
  • Mutagenesis
  • Protein Binding
  • Protein Stability
  • Protein Unfolding
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / metabolism*
  • Thermodynamics
  • Transition Temperature

Substances

  • Ligands
  • Proteins