Fluorophores covalent modification

Antibody molecules can be labeled with any one of more than a dozen different fluorescent probes currently available from commercial sources. Each probe option has its own characteristic spectral signals of excitation (or absorption) and emission (or fluorescence). Many derivatives of these fluorescent probes possess reactive functionalities convenient for covalently linking to antibodies and other molecules. Each of the main fluorophore families contains at least a few different choices in coupling chemistry to direct the modification reaction to selected functional groups on the molecule to be labeled. These choices include amine-reactive, sulfhydryl-reactive, and carbonyl-reactive. Examples of some of the more popular varieties of fluorescent probes can be found in Chapter 9. 

The GFP from the jellyfish Aequorea victoria, although not an enzyme, has become widely used as a marker for gene expression and localization. Although the fluorophore of GFP is not technically a protein-derived cofactor, it is a protein-derived fluorophore. This is another example of posttranslational modifications, which endow amino acid residues with a new function. In this case, the new function is not one which assists in catalysis. Instead, the results of these posttranslational modifications create new fluorescent properties, which serve a different biological function. As with most of the protein-derived cofactors discussed earlier, the presence and identity of the fluorophore is not evident from the amino acid sequence of the protein. The structure of the GFP fluorophore and mechanism of its biosynthesis were deduced from structural analyses. The X-ray crystal structure of GFP revealed that the covalently bound fluorescent chromophore is derived from three adjacent amino acids, serine-tyrosine-glycine on the polypeptide chain (Figure 13). ... 

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