| Photo-switching of protein functions has recently become more actively investigated by many researchers. Our objective is to create novel photo-switchable proteins by conjugating photo-responsive polymers to proteins site-specifically.; We synthesized N,N-dimethylacrylamide (DMA)-co-phenylazophenylacrylate copolymer (DMAA) and DMA-co-phenylazophenylacrylamide copolymer (DMAAm) with vinylsulfone termini for conjugation to a protein containing a cysteine. These polymers exhibited temperature-induced phase transitions, and opposite and reversible photo-responses; DMAA became soluble, while DMAAm became insoluble upon the same UV light irradiation.; The mutant streptavidins (SAs), E116C and S139C, were conjugated to these polymers site-specifically. The conjugates bound the ligand, biotin, below the phase transition temperature of the polymer, and released it above the phase transition temperature. The E116C-DMAA conjugate released biotin upon VIS light, while the E116C-DMAAm conjugate released it upon UV light. These opposite photo-responses corresponded to the opposite photo-induced phase transitions of the conjugated polymers. The E116C conjugate, which contains the conjugation site at a critical position for biotin-binding, exhibited larger responses than the S139C conjugate which contains it on the opposite side to biotin-binding. The conjugation of larger MW polymer and the addition of free polymer also increased the responses.; The mutant endoglucanaseIII, S25C and N55C, were also conjugated to the polymers. The conjugates exhibited similar temperature- and photo-responsive activity changes to the SA conjugates. Polymers with higher MW and the addition of the free polymer enhanced the switching activity. The N55C conjugates, which locate the conjugated polymer closer to the catalytic site than S25C, exhibited higher switching activity than the S25C conjugates. Both conjugates exhibited less activity toward a large substrate, hydroxyethylcellulose, than toward a small substrate, o-nitrophenylcellobioside. Kinetic studies revealed that the activity shut-off stemmed from the sterical inhibition by the shrunken polymers.; Therefore, the photo-switching of molecular recognition processes and catalytic activity were achieved by photo-responsive polymer-protein site-specific conjugation. The MW of the polymer, the position of conjugation, the size of the substrate and the addition of free polymer were key factors in the design of such molecular switches. This methodology can be utilized to develop novel photo-switchable proteins in the fields of biomedicine, bioprocesses, and bioelectronics. |
