Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

Abstract

Fluorescent labelling of specific proteins in complex biological systems remains an important challenge in chemical biology. One promising approach comprises the use of small molecules designed to react specifically with a targeted protein of interest and to increase in fluorescent intensity following this reaction. This kind of fluorogenic reaction generally derives from fluorescence quenching in the unreacted probe that is abrogated over the course of the reaction.Herein, we review the mechanistic principles of three major photophysical quenching mechanisms, involving Förster resonance energy transfer (FRET), through-bond energy transfer (TBET), and photoinduced electron transfer (PeT). We then present design principles for novel fluorogenic probes based on an understanding of these quench mechanisms, with emphasis on the emerging utility of density functional theory (DFT) calculations in the design process. DedicationThe authors dedicate this manuscript to Prof. R. S. Brown, the doctoral supervisor of JWK. Prof.Brown's acclaimed research continues to inspire the scientific community, notably through the rigour and depth of its mechanistic investigations, followed by its creative and relevant practical applications of that knowledge.

Bistetrazine‐Cyanines as Double‐Clicking Fluorogenic Two‐Point Binder or Crosslinker Probes

“…[1][2][3][4][5][6] In some cases,t hese fusiont ags may alter the original function of the protein of interest (POI) due to their comparable size. [17]). [7][8][9][10] In such labeling schemes, genetic incorporation of ab ioorthogonalized, noncanonical aminoa cid (ncAA) is followed by as pecific reactionw ith a small synthetically tailored dye.…”

A Green BODIPY‐Based, Super‐Fluorogenic, Protein‐Specific Labelling Agent

“…[11][12][13] As itespecific enzymatic reaction can then be used to affix af luorescent label to the POI. This method perturbs the POI less than the above-mentioned techniques,g iven the smaller size of the tag;h owever,n ative enzymatic reactions may prove problematic in some cellular applications.4 ) Finally,aPOI can be genetically modified to incorporate an unnatural amino acid, or fused to aminimal peptide tag that can react with small molecular probes, [14,15] avoiding the steric hindrance of large genetically fused proteins.…”

“…Among the small molecular agents used for the fluorescent labelling of specific proteins,t hose that are fluorogenic are advantageous relative to those that are always fluorescent. [15] TheF lAsH and ReAsH organoarsenic dyes developed by Tsien were among the first to be broadly applied, [16,17] despite their known cytotoxicity.O thers have been recently developed as fluorogenic versions of bioorthogonal labelling agents,i ncluding azidofluoresceins, [18] BODIPY-tetrazines [19] and fluorogenic inhibitors of enzyme-based SNAP-tag,Halo-Taga nd BL-tag. [20,21] We have developed ac omplementary strategy for covalent, fluorogenic protein labelling that relies on the quenching properties of maleimides and their specific fluorogenic addition reaction (FlARe) with as mall, genetically encoded dicysteine peptide tag.…”

A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation

“…[13] In contrast to other processes that result in quenched fluorescence through energy transfer, such as FRET, PET quenching does not require spectral overlap of the electron donor and acceptor and occurs over very short distances (≈ 3–5 Å). [14] Inspired by the use of conditional thioamide quenching for sensing protease activity [14a] and conformational dynamics of macromolecules, [14a,15] we have adapted this approach to observe the disorder-to-order transition of the 4E-BP1 “hot-spot” peptide. Herein, we describe the serendipitous discovery and development of a fluorescent peptide reporter that can detect modulation of the 4E-BP1 helix at nanomolar peptide concentrations.…”

“…[14a,15a,19] We chose to incorporate thio-leucine in place of Leu59, which is contained within the helix, and thioisoleucine in place of Ile53, which is N-terminal to the helix (Figure 4 A). Fmoc-protected thioamide amino acid precursors were synthesized as described [14a,20] and used in solidphase peptide synthesis. Whereas additional thioamide moieties did decrease the overall fluorescence of the peptides, the S/B was not significantly improved (Figure 4 B).…”