The green fluorescent protein (GFP) is a globular protein with a β-barrel structure, within which a chromophore forms spontaneously. It is built from the amino acid residues Ser56-Tyr66-Gly67, which cyclize spontaneously inside the barrel, and this chromophore is the source of strong fluorescence. In the blue fluorescent protein (BFP), the Tyr66 residue is replaced by histidine, which affects the photochemical properties of the protein. Histidine is an essential amino acid with an imidazole side chain, enabling functions unavailable to other amino acids, since it can switch between neutral and positively charged states under physiological conditions, allowing a variety of interactions. Using the copper-catalyzed reaction of hydrogen azide and L-propargylglicine, its analogue azahistidine can be prepared. This can be selectively incorporated into proteins in place of histidine. In the course of this thesis, we generated expression vectors containing the sequences for superfolder GFP (sfGFP) and superfolder BFP (sfBFP) using molecular cloning, synthesized azahistidine, and incorporated it into both proteins with the help of auxotrophic bacterial strains, while also preparing their natural variants. The fluorescence of proteins containing histidine or azahistidine residues was measured at different pH values. For sfGFP with histidine, we obtained an excitation peak at ~480 nm and an emission peak at ~510 nm. Fluorescence intensity increased with rising pH up to pH 9, after which it slightly decreased. Upon replacement of histidine residues with azahistidine, an additional excitation peak appeared at ~390 nm. This arose from the transition of the tyrosine residue in the chromophore from its deprotonated to its neutral form. Substitution of histidine residues with azahistidine influenced the spectrum despite sfGFP not containing histidine directly in the chromophore. His148 is located in close proximity to the chromophore, and its replacement most likely created a slightly more acidic environment, which affected fluorescence. For sfBFP with histidine residues, the peaks appeared at ~380 and 450 nm, with maximum fluorescence intensity at pH 8. However, replacing histidine residues with azahistidine drastically reduced or abolished fluorescence, since this time the substitution occurred in the chromophore itself.
|
