Fluorescent labeling, insertion site

The most critical step in these experiments is the choice of the site for fluorescent labeling. All labels, in particular however the large GFP variants, carry the risk of impairing the function and disposition of the labeled protein, and care must be taken to assure that indeed the labeled protein retains essential properties of the parent protein, such as cell surface expression, ligand binding, and signaling in the case of receptors. The choice of the insertion site is facilitated by structural information, and by knowledge about functional sites in these structures. 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

Figure 9.3.1 Ten amino acids were inserted at positions 1 and 2 in a pentapeptide. After acetylation the array of substrates was treated with the protease thermolysin. The liberated terminal amino groups were labeled with fluorescein isothiocyanate and the fluorescing spots detected with a scanning fluorescence microscope. The pentapeptide with amino acid 1 in both sites was obviously most easily hydrolyzed, followed by the ones with amino acid 1 in site 2 and amino acid 7 or 8 in site 1.

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