Detection of Dihydrofolate Reductase Conformational
Change by FRET Using Two Fluorescent Amino Acids
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Abstract
Two fluorescent amino
acids, including the novel fluorescent species
4-biphenyl-l-phenylalanine (<b>1</b>), 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 [<b>1</b> and l-(7-hydroxycoumarin-4-yl)ethylglycine
(<b>2</b>)] 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 (<b>1</b> at position 17; <b>2</b> at position 115) and DHFR II (<b>2</b> at position
17; <b>1</b> 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 <b>1</b>, energy transfer to <b>2</b>, and
emission by <b>2</b> 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