15N NMR studies of the conformation of E. coli dihydrofolate reductase in complex with folate or methotrexate.
We have employed 15N NMR to characterize the conformations of Escherichia coli dihydrofolate reductase (ECDHFR) in complex with [5-15N]folate or [5-15N]methotrexate (MTX). Two 15N resonances were observed for DHFR/MTX binary complex. The relative population of these two conformations is pH dependent. Addition of NADP+ or NADPH results in the disappearance of the low field resonance. In contrast, only one conformation was observed for both the DHFR/folate and DHFR/folate/NADP+ complexes. However, the 15N chemical shift of [5-15N]folate in the binary DHFR/folate complex is 7.28 ppm upfield from that of the ternary complex, suggesting the possible loss of a hydrogen bonding to N5 of folate in the ternary complex.
School of Physics, Georgia Institute of Technology, Atlanta 30332.
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D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000378:metabolism*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008727:Methotrexate / Q000378:metabolism*; D009587:Nitrogen Isotopes; D011485:Protein Binding; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
D009587:Nitrogen Isotopes; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate