2011
DOI: 10.1016/j.febslet.2011.10.014
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Identification of endogenous ligands bound to bacterially expressed human andE. colidihydrofolate reductase by 2D NMR

Abstract: Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and E. coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in t… Show more

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Cited by 14 publications
(19 citation statements)
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“…Frozen cell pellets can be stored for several weeks at −20 °C. hDHFR was purified and the desired complex was prepared by refolding as previously described 37 .…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…Frozen cell pellets can be stored for several weeks at −20 °C. hDHFR was purified and the desired complex was prepared by refolding as previously described 37 .…”
Section: Methodsmentioning
confidence: 99%
“…Affinity purification was carried out using NiNTA resin (Qiagen), and protein was eluted with 50 mM Tris pH 8.0, 200 mM NaCl, 1mM folic acid, 250 mM imidazole. Fractions containing DHFR were further purified by reversed-phase HPLC, and refolded previously described 37 . DHFR from each species was divided into two aliquots; one was refolded with NADP + and FOL, while the other was refolded with NADP + and THF.…”
Section: Methodsmentioning
confidence: 99%
“…Samples for assignments and 3 J coupling experiments were uniformly 13 C, 15 N-labeled and were purified as described previously. 25 NADPH and THF are light and air sensitive, and all samples containing these ligands were prepared in an argon-equilibrated glove box. The NMR buffer was thoroughly degassed under vacuum through a freeze-pump-thaw cycle and the protein was exchanged into NMR buffer [pH 7.6, 70 mM KPi, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 10% D 2 O].…”
Section: Methodsmentioning
confidence: 99%
“…In addition, this pathway does not exist in mammals; thus, the folate biosynthesis pathway has been considered a potential target during antibacterial compound development (31,32). In particular, folA is responsible for the reduction of dihydrofolate to tetrahydrofolate, which is crucial for protein and nucleic acid biosynthesis; folA is a well-studied target for the antibiotics trimethoprim and pyrimethamine (33)(34)(35)(36).…”
Section: Go Analysis Of the Unique Hits Of Each Amp-differentmentioning
confidence: 99%