Detection of dihydrofolate reductase conformational change by fret using two fluorescent amino acids

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

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Two fluorescent amino acids, including the novel fluorescent species 4-biphenyl-l-phenylalanine (1), have been incorporated at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes [1 and l-(7-hydroxycoumarin-4-yl)ethylglycine (2)] were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for Förster resonance energy transfer (FRET) measurements on the basis of their estimated separation (17-18 Å) and the expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II containing the potentially rotatable biphenylphenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of 1, energy transfer to 2, and emission by 2 at 450 nm. However, the energy transfer was substantially more efficient in DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration-dependent diminution of the emission observed at 450 nm for DHFR II but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function and in a fashion that enables sensitive monitoring of changes in DHFR conformation.

Original languageEnglish (US)
Pages (from-to)12924-12927
Number of pages4
JournalJournal of the American Chemical Society
Volume135
Issue number35
DOIs
StatePublished - Sep 4 2013

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Tetrahydrofolate Dehydrogenase
Amino acids
Amino Acids
Fluorophores
Energy Transfer
Energy transfer
Trimethoprim
Oxidoreductases
Phenylalanine
Proteins
Conformations
Irradiation
dihydrofolate reductase type II
Monitoring

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Detection of dihydrofolate reductase conformational change by fret using two fluorescent amino acids. / Chen, Shengxi; Fahmi, Nour Eddine; Wang, Lin; Bhattacharya, Chandrabali; Benkovic, Stephen J.; Hecht, Sidney.

In: Journal of the American Chemical Society, Vol. 135, No. 35, 04.09.2013, p. 12924-12927.

Research output: Contribution to journalArticle

Chen, Shengxi ; Fahmi, Nour Eddine ; Wang, Lin ; Bhattacharya, Chandrabali ; Benkovic, Stephen J. ; Hecht, Sidney. / Detection of dihydrofolate reductase conformational change by fret using two fluorescent amino acids. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 35. pp. 12924-12927.
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abstract = "Two fluorescent amino acids, including the novel fluorescent species 4-biphenyl-l-phenylalanine (1), have been incorporated at positions 17 and 115 of dihydrofolate reductase (DHFR) to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes [1 and l-(7-hydroxycoumarin-4-yl)ethylglycine (2)] were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for F{\"o}rster resonance energy transfer (FRET) measurements on the basis of their estimated separation (17-18 {\AA}) and the expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II containing the potentially rotatable biphenylphenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of 1, energy transfer to 2, and emission by 2 at 450 nm. However, the energy transfer was substantially more efficient in DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration-dependent diminution of the emission observed at 450 nm for DHFR II but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function and in a fashion that enables sensitive monitoring of changes in DHFR conformation.",
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