The Staudinger–Bertozzi ligation between an azide and a triarylph

The Staudinger–Bertozzi ligation between an azide and a triarylphosphine moiety ( Figure 1c) and alternatively the copper-catalysed [3 + 2] cycloaddition between an azide and an alkyne group [ 18] ( Figure 1a, also referred to as ‘click reaction’) are the most popular types of bioorthogonal reactions that can be used in vitro as well as in vivo because of their superior selectivity and biocompatibility [ 19]. Recently, copper-free click chemistry

has emerged that relies on strain-promoted cycloaddition making the reaction suitable for in vivo applications and work with highly sensitive protein learn more samples ( Figure 1b) [ 20]. In parallel, UAAs have been developed that can serve as reactant in a copper-free cycloaddition [ 21]. Plass et al. demonstrated that this approach leads to fluorescently labelled proteins suitable for single molecule studies [ 22•]. The Staudinger–Bertozzi ligation and cycloaddition can also be employed if the UAA carries the alkyne and the fluorophore is modified with the azide group, which is an attractive option because ERK inhibitor azides are often reduction-sensitive and labile during biochemical purification [ 23]. Many single molecule studies are designed to address the conformational flexibility of proteins in solution, or the structural organization either of

single proteins or protein complexes. Donor and acceptor probes for an intermolecular FRET system can be engineered into individual subunits that constitute a complex molecular Quisqualic acid machine or a heteromeric complex following standard coupling chemistries. In contrast, site-specific incorporation of donor and acceptor fluorophore in a single polypeptide is challenging and requires multiple unique coupling sites for differential labelling. A combination of the described coupling techniques often lead to successful dual labelling. For example, the N-terminus of a protein can be labelled via an amine-reactive group and a single cysteine with a thiol-reactive group.

Likewise, the modification of a single cysteine and an unnatural amino acid in a single protein chain is a sensible approach for an intramolecular site-specific labelling [23]. The incorporation of multiple [24] and two different UAAs [25] has been described, which opens the door for stochastic and site-specific labelling of proteins via the reactive side-chains of the UAAs. In some cases the site-specific positioning of the donor or acceptor probe is not mandatory to analyse the conformational flexibility or folding of a protein. Here, labelling via identical reactive moieties (e.g. 2 cysteines or 2 UAAs) is practicable [26]. Recently, advanced labelling strategies have been utilized to allow even triple-colour labelling within a single protein (stochastical labelling of two cysteines and one UAA) [27].

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