Determination of Dissociation Constants in Living Zebrafish Embryos with Single Wavelength Fluorescence Cross-Correlation Spectroscopy

Abstract

The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K(D)) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42(G12V) which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42(T17N). While Cdc42(G12V) binds to IQGAP1 with an apparent K(D) of approximately 100 nM, Cdc42(T17N) has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K(D) from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.

The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy

“…Fluorescence correlation spectroscopy (FCS) is widely used for measuring protein dynamics in live cells [1][2][3][4] and organisms [5][6][7][8]. FCS measures the dynamic properties of particles in a small laser focus by analyzing the emitted fluorescence fluctuations with a correlation analysis [9].…”

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

“…On the other hand, a few reports have measured dissociation constants in living cells (here referred to as the "in vivo K d ") (Fig. 1B) by means of intermolecular fluorescence resonance energy transfer (FRET), single-molecule fluorescence imaging, and fluorescence cross-correlation spectroscopy (FCCS) (9)(10)(11)(12). All of these techniques employ twocolor fluorescence imaging.…”

Protein-binding dynamics imaged in a living cell

“…Conversely, the cytoplasm of Escherichia coli was shown to destabilize the IgG binding domain of protein L (8). With respect to protein binding, it was shown that the affinity of Cdc42 to various proteins increases in eukaryotic cells (9), with a 10-fold difference between cell types (10). The dynamics of α 2A -adrenergic receptors binding to G proteins was monitored by Förster resonance energy transfer (FRET)-based assay, but no direct comparison to in vitro rate constants was made (11).…”