Quantifying protein-protein interactions in high throughput using protein domain microarrays

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein microarrays are effective for high-throughput protein-protein interaction studies.
• Recombinant protein production can be challenging due to diverse physicochemical properties.
• Protein interaction domains offer a more manageable approach.

Purpose of the Study:

• To develop protocols for constructing and utilizing protein domain microarrays.
• To quantify domain-peptide interactions efficiently.
• To assess binding selectivity across entire domain families.

Main Methods:

• Constructed microarrays of human Src homology 2 (SH2), phosphotyrosine binding (PTB), and mouse PDZ domains.
• Produced domains recombinantly in Escherichia coli.
• Quantified domain-peptide interactions using fluorescently labeled synthetic peptides and measured binding affinities (K(D)s) via saturation binding curves or fluorescence polarization.

Main Results:

• Successfully created domain microarrays for SH2, PTB, and PDZ domains.
• Enabled direct measurement of equilibrium dissociation constants (K(D)s) for high-affinity interactions.
• Facilitated identification and quantification of weaker interactions using fluorescence polarization.

Conclusions:

• Protein domain microarrays allow rapid, high-throughput identification and quantification of protein-ligand interactions with minimal sample usage.
• Interrogating entire domain families simultaneously provides a powerful tool for assessing binding selectivity.
• Offers an unbiased perspective on protein-protein interaction network connectivity.