Fluorescent dyes used for single molecule fluorescence applications commonly exhibit a maximum extinction coefficient ɛmax > 80.000 mol−1 cm−1 and a fluorescence quantum yield of Φ > 0.1. Their fluorescence lifetime is of the order of a few nanoseconds and their Raf activation size is roughly one nanometer. Bioconjugation is commonly carried out with fluorophore derivatives that target the functional side chains of specific native or engineered amino acids in a protein. The fluorophore attachment site has to be carefully chosen in order to prevent label-induced alteration of the protein’s activity and folding. The coupling reaction should be efficient in aqueous buffers at neutral pH and ambient

temperatures as most proteins Dapagliflozin datasheet are not soluble in organic solvents and tend to unfold or aggregate at high temperatures

and in highly basic or acidic environments. In addition, the coupling reaction needs to be highly chemoselective to ensure site-specific labelling of a single site in the protein. To this end coupling to amines and thiols are the most common labelling strategies that work efficiently under mild reaction conditions [10]. Newly developed technologies like bioorthogonal chemistry in combination with genetic engineering facilitate the site-specific labelling of unnatural amino acids (UAA) at any given position in a protein [11] improving the freedom of label positioning particularly in large proteins Urease hitherto inaccessible for site-specific labelling because of first, their high cysteine content, second, an unfavourable position of the cysteine residue in the core of the protein or third, the essential role of the cysteine in the coordination of bivalent metal ions as seen

in zinc-containing proteins. The coupling chemistries used in bioorthogonal reactions rely on unique chemical groups (e.g. para-acetyl or para-azide moieties) that are not part of the biological repertoire of amino acids [12• and 13]. However, several conditions have to be fulfilled to make such a strategy successful. The UAA — that is supplied to the growth media — has to cross the membrane of the bacteria and be compatible with the bacterial metabolism (i.e. not be cytotoxic). A unique amber stop codon (TAG) is engineered into the desired labelling site that serves as a coding codon for the unnatural amino acid. Plasmid-borne pairs of engineered orthogonal tRNAs and aminoacyl-tRNA synthetases facilitate the efficient loading of the UAA to the tRNA and subsequent incorporation of the UAA at amber stop codons. tRNA loading by the tRNA synthetase has to be highly specific for the exogenous amino acid but at the same time needs to be compatible with the bacterial translation machinery. Directed protein evolution schemes yielded several orthogonal pairs that have been adapted for use in Escherichia coli [ 14, 15 and 16].