title: Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics.
abstract: 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-tRNA(CUA). Excimer formation (λ(ex) 342 nm; λ(em) 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.
authors.affiliation: Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: Shengxi
authors.initials: S
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Nour Eddine
authors.initials: NE
authors.lastname: Fahmi
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D000409:Alanine