| Photoactive molecules are compounds whose chemical characteristics including bonds, charge, shape and polarity can be changed with appropriate wavelengths of light. There are two types of photoreactive moieties. The first change reversibly like azobenzene, which photoisomerizes around a central double bond, cycling between cis and trans conformers. The second irreversibly changes shape and includes o-nitrobenzyl caged compounds in which the caging group is photolyzed by 365 nm radiation. With the advent of unnatural amino acid mutagenesis, it has become possible to incorporate amino acids with a photoactive sidechain at any position in nearly any protein. The first photochromic amino acid incorporated this way was the azobenzene containing azophenylphenyl alanine (AzoPhe). cAMP receptor protein (CRP), a bacterial transcription factor activated by high cAMP levels was expressed in E. coli with AzoPhe at various positions. Using in vitro gel shift assays, two CRP AzoPhe mutants exhibited reversible changes in DNA-binding affinity upon illumination. With directed evolution, a synthetase for o-nitrobenzyl-O-tyrosine (ONBY) was also identified. This allowed photocontrol of protein function within living E. coli for the first time. beta-galactosidase with a catalytic tyrosine mutated to ONBY could be photoactivated in vivo or in vitro. In addition, ONBY synthesized from tyrosine bearing IR-visible C-D bonds can be selectively incorporated into proteins at any location with sire directed mutagenesis. Photolysis of the o-nitrobenzyl cage produces a wildtype protein with a single deuterated residue. With this strategy, it was possible to observe electrostatic coupling between DHFR active site residues and substrate analogs. In an effort to facilitate the photocontrol of mammalian cell processes, a pyrrolysine tRNA- tRNA synthetase pair was adapted for unnatural amino acid mutagenesis in both E. coli and mammalian cells. With this system, a synthetase for o-nitrobenzyloxycarbonyl-Nepsilon-lysine was evolved in E. coli and exported for mammalian genetic code expansion with caged lysine. With unnatural amino acid mutagenesis and photoactive proteins it has been possible to photocontrol biological activity in vitro , in the simple living system Escherischia Coli and in complex mammalian cells. |
