Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging

Abstract

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescent labeling with high efficiency, specificity, and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. We demonstrate that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in singlemolecule pull-down analysis. We further show the unique power of our method in a series of single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Sortase-mediated fluorescent labeling of CRISPR complexes

“…FGE turns the cysteine within the LCTPSR recognition motif into formylglycine. This residue can then be coupled to a cyanine hydrazide dye (Shi et al, 2012). While this labeling strategy can reach 100% labeling efficiency, it requires a large excess of fluorophore to label the protein (e.g., >75 mM in this reported study).…”

“…Using large concentrations of a fluorophore can be expensive and limited by the solubility of that particular dye. Alternatively, lower concentrations of fluorophores require a more acidic buffer and incubation at higher temperatures (e.g., pH 5.5 and 37°C) in order to get efficient labeling (Carrico, Carlson, & Bertozzi, 2007;Shi et al, 2012). These harsh conditions can cause unstable proteins to precipitate out of solution and limit the types of proteins that can be labeled via this method.…”

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria

“…It is well-known that the formyl group easily reacts with primary amines to form imine derivatives as Schiff bases [ 11 , 12 , 13 , 14 , 15 ]. For this reason, we developed a reactive ratiometric near-infrared fluorescent probe ( BH + ) for pH detection in mitochondria by attaching a formyl group to a hemicyanine dye in order to prevent the probe from diffusing away from mitochondria.…”