Fluorescent Protein Engineering Through Genetic Incorporation of 3-Chlorotyrosine

Posttranslational chlorination of tyrosine residues in proteins produce 3-chlorotyrosine (3-Cl-Tyr), which is associated with several diseases, including Alzheimer's disease, asthma, atherosclerosis and acute myocardial infarction. High level of 3-chlorotyrosine has been found in ApoA1 protein in atherosclerosis patients, indicating that it may play important role in disease. Here we report a new method to facilitate the site-specific incorporation of 3-chlorotyrosine into proteins at specific sites. Such a new method may be very useful to probe the regulatory role of tyrosine chlorination in protein function. Compared to tyrosine (Tyr), 3-Cl-Tyr has lower pK(a). We replaced the green fluorescent protein (GFP) and photoactivatable protein mEOS2 chromophore Tyr (Tyr66 in GFP) by 3-Cl-Tyr, lowering the chromophore pK(a) to 4.2 and 4.7, respectively. These mutant fluorescent proteins with lower pK(a) may be advantageous for labeling proteins in acidic organelles such as lysosome and phagosome.