Minimalist Approaches to Protein Labelling: Getting the Most Fluorescent Bang for Your Steric Buck

Abstract

Fluorescence methods allow one to monitor protein conformational changes, protein–protein associations, and proteolysis in real time, at the single molecule level and in living cells. The information gained in such experiments is a function of the spectroscopic techniques used and the strategic placement of fluorophore labels within the protein structure. There is often a trade-off between size and utility for fluorophores, whereby large size can be disruptive to the protein’s fold or function, but valuable characteristics, such as visible wavelength absorption and emission or brightness, require sizable chromophores. Three major types of fluorophore readouts are commonly used: (1) Förster resonance energy transfer (FRET); (2) photoinduced electron transfer (PET); and (3) environmental sensitivity. This review focuses on those probes small enough to be incorporated into proteins during ribosomal translation, which allows the probes to be placed on the interiors of proteins as they are folded during synthesis. The most broadly useful method for doing so is site-specific unnatural amino acid (UAA) mutagenesis. We discuss the use of UAA probes in applications relying on FRET, PET, and environmental sensitivity. We also briefly review other methods of protein labelling and compare their relative merits to UAA mutagenesis. Finally, we discuss small probes that have thus far been used only in synthetic peptides, but which have unusual value and may be candidates for incorporation using UAA methods.

Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection

“…9 To appropriately model protein motions, one needs a set of probes that are capable of accurately reporting on distance changes without disrupting the fold and function of the protein of interest. 10 …”

A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis elegans

“…For example, the majority of fluorogenic probes published to date target enzymes 4,7,14,18–24 . It would be ideal if fluorogenic probes could be routinely developed for non-enzyme proteins and other macromolecules 8,13,15,16,25–30 . Moreover, a better understanding of the physicochemical underpinnings of fluorogenicity would enable more efficient design 18,31,32 .…”

Multiply labeling proteins for studies of folding and stability

“…[7] However, protein tags can be perturbing to the processes being studied, owing to the size of the label relative to the protein of interest. [8,9] Smaller label strategies include fluorogenic bisarsenical dyes that are selectively chelated by tetracysteine motifs and chemoenzymatic labeling using enzymatic recognition of short peptide sequences. [10-12] Such methods rely on insertion of a 5-15 amino acid sequence, which may still be disruptive to native protein structure and function.…”