A novel immobilization method for single protein spFRET studies

Abstract

We have developed a new method for immobilization of single proteins by utilizing streptavidin-biotin and protein L-antibody interactions on glass coverslips coated with polyethylene glycol. The method is particularly well suited for single-molecule fluorescence studies. A monomeric, detergent-solubilized bacterial transport protein, GlpT, and the dimeric cytoplasmic region of a mammalian transporter, cdAE1, were immobilized by our method with a high degree of specificity. The fluorescence from single molecules attached to the immobilized proteins was detected with a high signal/noise ratio. Single-pair fluorescence resonance energy transfer (spFRET) measurements on cdAE1 dimers indicate that the structure of the protein is not compromised and provide evidence that the cdAE1 protein can exist in at least two conformations under physiological conditions.

FIGURE 1

Schematic representation of the immobilization scheme (see Supplemental Materials for protocols).

FIGURE 2

(A) Raster-scanned fluorescence images of immobilized Cy3-GlpT (a 532-nm laser was used for excitation); inset shows fluorescence intensity at the scan region marked by the arrow. (B) Control experiment with no protein L-biotin.

FIGURE 3

(A) Simultaneous time-series recording of donor and acceptor channels and corresponding energy transfer efficiency. (B) Distribution of R/R₀. (C) Crystal structure of cdAE1 dimer showing Cys-201 locations as red dots (image created with PyMol).