Computational protocol: The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

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Protocol publication

[…] Rates of H/D exchange (HDX) at 20 °C were determined by NMR on a Varian INOVA 900 MHz (1H) spectrometer equipped with a cryogenically cooled HCN probe. Proteins for NMR experiments were purified by anion exchange on Q-sepharose resin followed by gel filtration on a prep grade Superdex 75 column. This ensures that no folate is present in the apoenzyme prior to NMR measurements, which would be the case using our usual purification method. EcDHFR and MpDHFR, as the apoenzyme and the NADP+/folate complexes (6-fold excess of ligands), were prepared as 2 mM stocks in 50 mM potassium phosphate buffer, pH 7, containing 1 mM NaCl, 10 mM β-mercaptoethanol, and 10% D2O. As MpDHFR cannot be freeze-dried without substantial loss of activity, HDX experiments were performed by dilution rather than resuspension. The concentrated stock was diluted 10-fold into H2O and D2O 24 h prior to measurement to create ‘before’ and ‘after’ samples, which were used to optimize the acquisition parameters and to obtain optimized shim maps for HDX experiments. HDX was then performed by diluting the concentrated stock 10-fold into D2O immediately prior to insertion into the magnet. The time between sample mixing and acquisition of the first spectrum was recorded. SOFAST-HMQC spectra were recorded at regular intervals until no further exchange was seen, increasing the number of scans with time to maximize signal (see for full details). Spectra were processed using NMRPipe and analyzed using Analysis 2.1.5 for Linux. Peak intensity (adjusted for changes to the number of scans by dividing by the square root of the ratio of number of scans) was plotted against exchange time (from sample mixing to the midpoint of the spectrum acquisition) and rate constants determined using SigmaPlot 10.1H–15N crosspeaks for the EcDHFR/NADP+/folate complex were assigned using published data, and connectivity was confirmed using a 3D 1H–15N NOESY-HSQC spectrum acquired on a Varian INOVA 600 MHz (1H) spectrometer equipped with a HCN probe, using 2.4 mM EcDHFR with 15 mM ligands in 50 mM potassium phosphate buffer, pH 7, containing 1 mM NaCl, 10 mM β-mercaptoethanol, and 10% D2O. Crosspeaks for apo-EcDHFR were assigned by titration to form the NADP+/folate complex and using a 3D 1H–15N HSQC-NOESY spectrum acquired on a Bruker Avance II+ 700 MHz (1H) spectrometer equipped with a cryogenically cooled HCN probe, using 2.2 mM apo-EcDHFR in the same buffer. Crosspeaks for the MpDHFR/NADP+/folate complex were assigned using standard triple-resonance methods. As all apo-MpDHFR crosspeaks exchanged fully within the dead time of the experiment, no assignment was performed.1H–15N HSQC spectra were also acquired for the EcDHFR/NADP+/folate complex in the presence of 17, 33, and 50% methanol and glycerol. Spectra were acquired on a Varian INOVA 600 MHz (1H) spectrometer equipped with a HCN probe, using 240 μM EcDHFR with 1.5 mM ligands in 50 mM potassium phosphate buffer, pH 7, containing 1 mM NaCl, 10 mM β-mercaptoethanol, and 10% D2O. […]

Pipeline specifications

Software tools NMRPipe, SigmaPlot
Applications Miscellaneous, NMR-based metabolomics
Organisms Escherichia coli