Microsecond Time Scale Dynamics in Proteins A R T I C L E S Figure 1. Experimental schemes used to measure 1H relaxation dispersion proﬁles of CHD2 methyl groups. microsecond time scale exchange processes, which is crucial to adequately map the chemical exchange spectral density function. Materials and Methods Sample Preparation. The heat- and IPTG-inducible pRCB1 expression vector, containing the P43G mutation, was used to obtain the coding sequence for calbindin D9k by PCR using 5′-ggctcAT- GAAATCTCCGGAAGAACTG and 5′-cgctcatatggaTTTCTG- GCAGGG (uppercase, annealing sequence; lowercase, primer overhang) as the forward and reverse primers, respectively. The PCR product was puriﬁed from agarose gel, cut with PagI/NdeI and ligated into the NcoI/NdeI digested pET-15b vector (Novagen). The resulting pET15b-calbindinP43G expression vector (IPTG- inducible, no His-tag) was sequenced (Service XS, Leiden, The Netherlands) and conﬁrmed to be correct. Escherichia coli BL21(DE3) was used to express recombinant calbindin D9k. For the induction of protein synthesis, cells were grown at 37 °C in M9 minimal medium with 100 µ g/mL ampicillin to an OD600 ≈ 0.6 and incubated for 3 h at the same temperature in the presence of 1 mM isopropyl -D-thiogalactoside (IPTG). To purify calbindin D9k, cell pellets were resuspended in a buffer containing 20 mM Tris, pH 8.0 (buffer A) with 10 µ g/mL DNase I and 0.5 mM phenylmethanesulfonyl ﬂuoride (PMSF) added. After French press, the cell lysate was added to twice the volume of boiling buffer A, and under vigorous stirring it was heated until the temperature reached ∼95 °C. After quickly cooling the sample on ice-water slurry, the precipitated proteins and cell debris were removed by centrifugation. The supernatant was loaded onto a Q-Sepharose column (GE Healthcare) pre-equilibrated with buffer A. After extensive washing with buffer A, calbindin D9k was eluted using a salt gradient of NaCl (0-200 mM). Fractions containing calbindin D9k were pooled, dialyzed extensively against water, and the homogeneity was conﬁrmed by 15% SDS-PAGE. A U-[13C, 15N]-1H/2H sample of calbindin D9k was obtained using the general isotope labeling strategy outlined by Tugarinov et al.76 Brieﬂy, pET15b-calbindinP43G was transformed into E. coli BL21(DE3) and was grown in 200 mL of D2O M9 medium containing 2 g/L U-[1H, 13C]-D-glucose (Cambridge Isotope Labo- ratories) and 1 g/L 15NH4Cl (Cambridge Isotope Laboratories) as the sole carbon and nitrogen sources, respectively. The calbindin D9k (P43G) NMR sample contained ∼1 mM protein, pH 6.0 (93% H2O and 7% D2O) and was fully calcium-loaded. NtrCr was prepared and puriﬁed as described previously,71 except that in this case [U-1H,1-13C]-D-glucose was used as the sole carbon source and bacterial growth was performed in ∼100% D2O. The NtrCr NMR sample contained ∼0.75 mM protein and 50 mM sodium phosphate, 0.02% azide, pH 6.75 (90% H2O and 10% D2O). NMR Spectroscopy. NMR experiments were performed on Varian Unity Inova spectrometers operating at 600 and 800 MHz, equipped with a triple-resonance room temperature (600 MHz) or a cryogenically cooled (800 MHz) probehead. Relaxation dispersion experiments on calbindin D9k were re- corded at 28 °C on a 600 MHz spectrometer with the experimental schemes A and B (Figure 1) as a series of 25 2D data sets with TCP set to 80 ms and the constant-time (CT) carbon evolution period equal to 28 ms. The CPMG ﬁeld strengths, νCPMG (νCPMG ) 1/(4τc), where 2τc is the interval between the 180° proton pulses during the CPMG element40), were equal to 25, 50, 75, 100, 200, 325, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, 2150, 2200, 2300, and 2500 Hz with duplicate experiments recorded at 100, 400, 800, and 1600 Hz. For the experiment performed using scheme C (Figure 1), a series of 23 2D data sets was recorded with CPMG ﬁeld strengths, νCPMG, equal to 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 675, 750, 800, 900, 950, and 1000 Hz with duplicate experiments recorded at 100, 200, 400, 800, and 1000 Hz. The 13C CW decoupling ﬁeld was centered at 25 ppm and had a ﬁeld strength, ω1/2π, varying between 1.2 and 2.1 kHz, following the procedure described by Vallurupalli et al.77 The 13C B1 ﬁeld was calibrated using the method described by Mulder and Akke63 from data recorded on the same protein sample. The reference experiment was performed by omitting the CPMG block in the pulse scheme and repeated two and four times for schemes A/B and C, respectively. The CHD2-detected 1H-CPMG experiments were acquired with 111 (13C) × 512 (1H) complex points, with maximum evolution times equal to 27.75 (13C) × 64 (1H) ms. An interscan delay of 1.5 s was used with four scans per transient, giving rise to a net acquisition time of ∼25 min per 2D spectrum. Relaxation dispersion experiments on NtrCr were recorded at 25 °C with the experimental scheme D (Figure 1) at two static (76) Tugarinov, V.; Kanelis, V.; Kay, L. E. Nat. Protoc. 2006, 1, 749– 754. (77) Vallurupalli, P.; Scott, L.; Williamson, J. R.; Kay, L. E. J. Biomol. NMR 2007, 38, 41–46. J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 17007