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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

Watson, MD; Monroe, J; Raleigh, DP; (2018) 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. Journal of Physical Chemistry B , 122 (21) pp. 5278-5285. 10.1021/acs.jpcb.7b05684. Green open access

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Abstract

Changes in protein stability are commonly reported as changes in the melting temperature, ΔTM, 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 ΔTM. A linear relationship is observed for each protein. The slopes of the plots of ΔTM 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 TM 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 TM. The relationship between ΔΔG° and ΔTM 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, ΔTM, scales as N–1. Hence, thermal shift assays are considerably less sensitive for ligand binding to larger proteins.

Type: Article
Title: 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
Open access status: An open access version is available from UCL Discovery
DOI: 10.1021/acs.jpcb.7b05684
Publisher version: http://doi.org/10.1021/acs.jpcb.7b05684
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: https://discovery.ucl.ac.uk/id/eprint/10053599
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