Two Pyrenylalanines in Dihydrofolate Reductase Form an Excimer Enabling the Study of Protein Dynamics
2012
Chen, Shengxi | Wang, Lin | Fahmi, Nour Eddine | Benkovic, Stephen J. | Hecht, Sidney M.
Because of the lack of sensitivity to small changes in distance by available FRET pairs (a constraint imposed by the dimensions of the enzyme), a DHFR containing two pyrene moieties was prepared to enable the observation of excimer formation. Pyren-1-ylalanine was introduced into DHFR positions 16 and 49 using an in vitro expression system in the presence of pyren-1-ylalanyl-tRNACUA. Excimer formation (λₑₓ 342 nm; λₑₘ 481 nm) was observed in the modified DHFR, which retained its catalytic competence and was studied under multiple and single turnover conditions. The excimer appeared to follow a protein conformational change after the H transfer involving the relative position and orientation of the pyrene moieties and is likely associated with product dissociation.
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