Quantifying protein interactions in a living organism

In recent years, experiments have shown that weak protein-protein interactions are influenced by enthalpic ('sticking') and entropic ('crowding') effects of their environment. The chaperoning activity of the molecular chaperone Hsp70 and its client Phosphoglycerate Kinase (PGK) lies in the realm of weak protein-protein interactions. In this work, we demonstrate a pipeline to study such protein interactions in different tissues of live zebrafish larvae. Using meganuclease-mediated transformation, we induce mosaic bicistronic expression of fluorescently tagged HspA1A variant of Hsp70 and a low melting point mutant of yeast PGK. We subject anesthetized larvae to a heat shock to induce measurable chaperoning interactions in its transformed cells. Using fluorescence resonance energy transfer (FRET), we detect the onset of binding of the two proteins near the melting temperature of PGK in the larval myocytes. Our experiments aim at quantitatively comparing these interactions in different tissues of the larvae as well as comparing the chaperoning activity of Hsp70 and the constitutively expressed homolog, heat shock cognate protein Hsc70 (HSPA8).