Expanding the Chemical Biologists Tool Kit: Chemical Labelling Strategies and Its Applications

Abstract

Methods that allow visualisation of proteins in living systems, in real time have been key to our understanding of the molecular underpinnings of life. Although the use of genetically encoded fusions to fluorescent proteins have greatly advanced such studies, the large size of these tags and their ability to perturb protein activity has been major limitations. Attempts to circumvent these issues have seen the genesis of complementary strategies to chemically label/modify proteins. Thus, chemical labelling approaches seek to "decorate" biomolecules in live cells through the site-specific introduction of a small, non-native chemical tag (or reporter group). The introduced tag is minimally invasive such that the activity and/or function of the target molecule in not perturbed/compromised by its inclusion. In most cases, this modification is brought about by fusing target biomolecules to protein domains/peptide tags or via the incorporation of reactive "handles" by either exploiting the cell's biosynthetic machinery or during protein synthesis. Selective tagging of the biomolecule then proceeds via a bioorthogonal chemical reaction following exogenous addition of probe(s). Depending on the nature of the probe, the method can be applied to either visualise/track the dynamics of target molecule(s) in their native cellular milieu or for affinity enrichment for further downstream applications. The versatility of these approaches has been demonstrated by their ability to tag not just proteins but also intractable biomolecules like lipids and glycans. In this review, we summarise the various strategies available to "chemically" tag proteins and provide a comparative analysis their advantages and disadvantages. We also highlight the many creative applications of such methodologies and discuss their future prospects.

Enzyme immobilization: an update

“…However, in order to exploit this attachment for protein immobilization, the protein of interest must first be labeled with biotin. Classically, this can be achieved with a number of nonselective chemical biotinylation reagents such as biotin NHS ester [36]. One conceptually straightforward method of protein immobilization is through the use of enzymatically active fusion proteins.…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…For quantitative FRET the microenvironment dependency of the fluorophore's photophysical properties and the uncertainty in probe position and orientation relative to the biomolecule should be considered when choosing donor/acceptor probes and bioconjugation strategies [6]. Donor/acceptor probe attachment to a biomolecule can be achieved via a number of labeling techniques that can be chemically or biologically inspired in nature (Figure 6.1); for recent reviews see Refs [10][11][12].…”

“…There are an expanding number of biologically inspired techniques that harness genetically encoded peptide handles combined with enzymes to catalyze small-molecule (e.g., biotin and fluorescent dye) conjugation [10][11][12]16,23,28,29]. Ref.…”

“…The HaloTag TM [haloalkane dehalogenase (DhaA) ] and SNAP TM -tag [O 6 -alkylguanine-DNA alkyltransferase (hAGT)] are labeling techniques that actually involve fusing the full enzymes (which self-label themselves) to the protein of interest. The HaloTag utilizes fluorescently labeled haloalkane substrates, while the SNAP-tag uses fluorescent benzylguanine derivatives to generate fluorescently labeled targets [10][11][12].…”

Fluorescent-tagged sp2-iminosugars with potent β-glucosidase inhibitory activity

“…Coupling of commercially available activated fluorescent probes to an aminefunctionalized scaffold was selected as the conjugation method of choice since it represents one of the most efficient and popular strategies for the preparation of fluorescent-tagged conjugates. 20 Moreover, this approach is well-suited for convergent schemes in which the fluorescent moiety is incorporated at the very last step. Following these considerations the 5N,6S-(4 0 -aminobutyliminomethylidene)-6-thionojirimycin derivative 10 (Scheme 1) was conceived as the key pivotal intermediate for the preparation of the title compounds.…”