A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes

Abstract

Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar Kd values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.

Chart 1

Structures of fluorogenic cyanine dyes.

Figure 1

Fluorescence titration curve for binding of DIR to surface-displayed clone K7. Line represents fit to 1:1 binding model. Open circles and filled triangles: titration of dye into cells not expressing the scFv and buffer, respectively. Inset shows calculated K_d values for five unique clones selected by flow cytometry.

Figure 2

Scanning laser confocal fluorescence image of yeast expressing FAP K7 in the presence of 100 nM DIR.

Figure 3

Dissociation constants and normalized fluorescence spectra for various unsymmetrical cyanines bound to scFv clone K7.