Exploiting Binding Kinetics of Fluorogen Activating Peptides to Enhance Photostability: Applications to Live Cell Single Molecule Imaging

Abstract

Inefficiency of fluorescent labels imposes key limitations in fluorescence microscopy. Photobleaching, non-specific signal from multiple fluorophores, and lack of multiplexing and orthogonal labels impose major challenges in studying complex cellular systems, both at ensemble and single molecule levels. We have addressed these problems by exploiting the binding kinetics of fluorogen activating peptides that on binding to a non-fluorescent molecule (a fluorogen) exhibit fluorescence. Novel methods in flow cytometry and rational design of peptides enabled the elucidation of peptides and fluorogen pairs with varying off rates. Complexes with fast off rates (koff∼5E-3 s-1) can exchange a bleached fluorogen molecule with a fresh fluorogen molecule in solution. Since the free fluorogen is dark, we can image for extended time scales in the presence of excess fluorogen. For complexes with slow off rates (koff∼5E-5 s−1) and high affinity (Kd<1E-9M), we can target commercially available Streptavidin QDots to proteins on the cell membrane, cytoplasm and the nucleus. We have imaged these proteins at a single molecule level for long time scales by exploiting the exceptional photostability of QDots. We have shown the use of this technique in particular, to the study of the diffusion dynamics of the β-2 adrenergic receptor and a trans-membrane protein on the cell surface simultaneously in real time. We have also developed peptides that non-covalently target cyanine dyes with sub-nanomolar affinities. This solves the problems associated with protein-dye conjugation and enhances the photostability of these dyes up to ten fold. We have demonstrated the use of these peptides in single molecule studies of membrane proteins. Thus by exploiting kinetics, we have developed a genetically encoded toolbox for targeting fluorogens, organic dyes and QDots for the study of proteins on and inside the cell.