Abstract
Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (KD) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for inner-ear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (Tm) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These Tm shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the Tm shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)- pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly two-fold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (KD from nM up to 100 μM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.
Highlights
Protein-protein interactions are intrinsic to virtually every cellular process whether it be transcription, translation, replication, cell cycle control or signal transduction [1,2,3]
Using thermal scanning with SYPRO orange to detect unfolding, we found that pcdh15 melts at lower temperatures than cdh23, and that the apparent melting temperature of pcdh15 increases in the presence of cdh23
To study protein-protein interactions in a high-throughput fashion, we set out to investigate the application of thermal scanning assays using the CDH23-PCDH15 complex
Summary
Protein-protein interactions are intrinsic to virtually every cellular process whether it be transcription, translation, replication, cell cycle control or signal transduction [1,2,3]. These interactions are essential for multicellular organisms in processes such as cell-cell adhesion, hostpathogen interactions, and sensory perception [4,5,6]. Measurements of binding affinity and kinetic rate constants are often used along with site-directed mutagenesis to understand the molecular mechanisms underlying protein-protein interactions. The quantitative determination of dissociation constants (KD) can be done in multiple ways, e.g., by measuring association-.
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