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J Am Chem Soc. 2012 Nov 21;134(46):18883-5. doi: 10.1021/ja307179q. Epub 2012 Nov 8.

Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics.

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1
Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.

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.

PMID:
23116258
PMCID:
PMC3546169
DOI:
10.1021/ja307179q
[Indexed for MEDLINE]
Free PMC Article
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