Quantitation of protein-protein interactions by thermal stability shift analysis.


Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity- and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (∼ 100 nM to ∼ 100 μM).

Submission Details

ID: 35E8QAAe

Submitter: Shu-Ching Ou

Submission Date: Nov. 14, 2018, 11:23 a.m.

Version: 1

Publication Details
Layton CJ;Hellinga HW,Protein Sci (2011) Quantitation of protein-protein interactions by thermal stability shift analysis. PMID:21674662
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.7 Maltose/maltodextrin-binding periplasmic protein P0AEY0 MALE_ECO57
99.7 Maltose/maltodextrin-binding periplasmic protein P0AEX9 MALE_ECOLI
94.0 Maltose/maltodextrin-binding periplasmic protein P19576 MALE_SALTY
93.4 Maltose/maltodextrin-binding periplasmic protein P18815 MALE_KLEAE