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

Shengxi Chen; Lin Wang; Nour Eddine Fahmi; Stephen J. Benkovic; Sidney M. Hecht

doi:10.1021/ja307179q

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

Shengxi Chen, Lin Wang, Nour Eddine Fahmi, Stephen J. Benkovic, Sidney M. Hecht

Chemistry

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32 Scopus citations

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.

Original language	English (US)
Pages (from-to)	18883-18885
Number of pages	3
Journal	Journal of the American Chemical Society
Volume	134
Issue number	46
DOIs	https://doi.org/10.1021/ja307179q
State	Published - Nov 21 2012

All Science Journal Classification (ASJC) codes

Catalysis
General Chemistry
Biochemistry
Colloid and Surface Chemistry

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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- 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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T1 - Two pyrenylalanines in dihydrofolate reductase form an excimer enabling the study of protein dynamics

AU - Chen, Shengxi

AU - Wang, Lin

AU - Fahmi, Nour Eddine

AU - Benkovic, Stephen J.

AU - Hecht, Sidney M.

PY - 2012/11/21

Y1 - 2012/11/21

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

Abstract

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