1H, 13C and 15N Chemical Shift Assignments of Dihydrofolate Reductase from The

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1H, 13C and 15N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP+ and folate

E. Joel Loveridge, Stella M. Matthews,Christopher Williams, Sara B.-M. Whittaker, Ulrich L. Günther, Rhiannon M. Evans, William M. Dawson, Matthew P. Crump, Rudolf K. Allemann

Figure S1. Enlarged 2D 1H-15N-HSQC spectrum of 1.1 mM uniformly 15N-labelled MpDHFR in the presence of 11 mM each NADP+ and folate in 50 mM potassium phosphate buffer (pH 7.0) containing 100 mM NaCl, 10 mM 2-mercaptoethanol (90% H2O/10% D2O) and saturated with argon. The spectrum was acquired at 291 K on a Varian VNMRS 600 MHz spectrometer equipped with a cryogenically cooled probe. The assignments are labeled by the one-letter code of amino acids accompanied by a sequence number. The side-chain resonances of asparagine and glutamine residues are connected by horizontal lines.

Figure S2. Chemical shift differences between MpDHFR and EcDHFR. Blue symbol represent conserved residues; red symbols represent non-conserved residues.

Figure S3 (next page). 1H-15N HSQC spectra of MpDHFR as the (A) NADP+:folate ternary complex, (B) folate binary complex, (C) dihydrofolate binary complex, (D) NADP+:dihydrofolate ternary complex (prepared by adding equimolar NADP+ to the dihydrofolate binary complex), (E) NADPH binary complex, (F) NADP+:tetrahydrofolate ternary complex (prepared by adding equimolar dihydrofolate to the NADPH binary complex), (G) apo-enzyme and (H) NADP+:folate ternary complex. Spectra were measured using 1.1 mM MpDHFR with 11 mM ligands at 600 MHz (1H) and 291 K. MpDHFR for the spectra in panels A-G was prepared using argon-saturated buffers; MpDHFR for the spectrum in panel H was prepared without use of argon.

Figure S4. Enlargementsof the 2D 1H-15N-HSQC spectrum of MpDHFR prepared without the use of argon-saturated buffers(shown in panel H of Figure S2) demonstrating multiple resonances for some residues. The assignments are labeled by the one-letter code of amino acids accompanied by a sequence number.

Figure S5. (a) Cartoon representation of MpDHFR showing regions where multiple resonances are seen in the 1H-15N-HSQC spectrum when the protein is prepared without the use of argon-saturated buffers (red). Ligands are shown as sticks. (b) Slice though the HNCA (red), HN(CO)CA (green), HNCACB (blue) and CBCA(CO)NH (orange) spectra of MpDHFR at 15N 113.75 ppm showing the splitting of the Cys105 resonances due to the presence of the oxidized and reduced forms. For the oxidized side chain, the chemical shifts are N 113.71 ± 0.07 ppm, HN 7.90 ± 0.00 ppm, CA 55.56 ± 0.14 ppm and CB 43.73 ± 0.13 ppm. For the reduced side chain, the chemical shifts are N 113.77 ± 0.09 ppm, HN 7.81 ± 0.01 ppm, CA 60.70 ± 0.06 ppm and CB 30.35 ± 0.21 ppm.