Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Protein-protein interactions (PPIs) are fundamental to cellular processes.
• Accurate quantification of PPIs, especially low-affinity ones, remains a challenge.
• Existing methods often lack sensitivity or require purified proteins.

Purpose of the Study:

• To develop an ultrasensitive technique for quantitative protein-protein interaction analysis.
• To enable the study of PPIs directly from complex biological samples like cell lysates.
• To investigate the dimerization of signal transducer and activator of transcription (STAT) proteins and epidermal growth factor receptor (EGFR).

Main Methods:

• Utilizing phase-separated, micropatterned membranes for a two-dimensional assay format.
• Employing lipid probes and affinity tags for protein capture and interaction-induced partitioning.
• Quantifying interactions via fluorescence imaging of protein partitioning into the liquid-ordered phase.
• Calibrating the assay using well-defined low-affinity protein-protein interactions.

Main Results:

• Successfully quantified equilibrium dissociation constants greater than 1 mM.
• Detected homo- and heterodimerization of signal transducer and activator of transcription (STAT) proteins directly from mammalian cell lysates.
• Quantified low-affinity interactions between different domains of the epidermal growth factor receptor (EGFR) involved in dimerization.
• Demonstrated the regulation of EGFR dimerization by lipids using the developed assay.

Conclusions:

• The developed membrane-based assay provides an ultrasensitive platform for quantitative PPI analysis.
• The technique is applicable to complex biological samples, enabling in situ studies.
• This method offers new insights into the mechanisms of protein dimerization and its regulation, with implications for understanding cellular signaling pathways and disease.