A R T I C L E S magnetic ﬁelds, as a series of 55 (600 MHz) and 46 (800 MHz) 2D data sets, respectively, with TCP set to 40 ms and the constant- time (CT) carbon evolution period equal to 28 ms. The CPMG ﬁeld strengths, νCPMG, varied between 50 and 6000 Hz and included 18 (600 MHz) and 15 (800 MHz) duplicate points (see the Supporting Information for details, Table S1). The reference experiment was performed by omitting the CPMG block in the pulse scheme and was repeated four (600 MHz) or two (800 MHz) times. The CHD2-detected 1H-CPMG experiments were acquired with 111 (13C) × 512 (1H) complex points and maximum evolution times equal to 27.75 (13C) × 64 (1H) ms (600 MHz) or 140 (13C) × 640 (1H) complex points and maximum evolution times equal to 28 (13C) × 64 (1H) ms (800 MHz). An interscan delay of 1.5 s was used with 12 (600 MHz) or 4 (800 MHz) scans per transient, giving rise to a net acquisition time of 1.25 h (600 MHz) or 31.5 min (800 MHz) per 2D spectrum. The heating of the protein sample due to the CPMG block in the pulse scheme was determined by comparison of the 1H chemical shifts78,79 between an experiment with and without the CPMG scheme and found to be 0.4 and 1.0 °C for calbindin D9k and NtrCr, respectively. The setting of the variable temperature (VT) unit was adjusted such that the “real” sample temperature was 28 °C for calbindin D9k and 25 °C for NtrCr. To ensure that the sample heating remains constant for each of the different experimental schemes and for different values of νCPMG, a period TCP was added at the beginning of the interscan delay, d1, such that the total number of 180° proton pulses was maintained. Pulse Sequence. The experimental schemes for the sensitivity- enhanced 1H-CPMG-CHD2 experiment are shown in Figure 1. The core of the experiment is a CT-[1H-13C]-HSQC pulse sequence to detect only the CHD2 isotopomer as described earlier.58 The CPMG part is implemented during the ﬁrst INEPT period as a variable pulse spacing,23,24 constant-time (CT) CPMG element,40 either as relaxation-compensated variant29 (rcCPMG; Figure 1, scheme A) or applied to the in-phase magnetization with 13C CW decoupling (cwCPMG; Figure 1, scheme B). Schemes C and D can be used to substitute for the conventional CT carbon evolution period. The individual schemes and their beneﬁt for different experimental conditions and isotopically labeled protein samples are described in more detail in the Results and Discussion and in the Supporting Information. Narrow (wide) ﬁlled bars indicate 90° (180°) RF pulses applied along the x-axis, unless otherwise indicated. The proton pulse train for heat-compensation at the beginning of the sequence (not shown in Figure 1) is performed 200 kHz off-resonance, and the 1H carrier is moved to the water resonance (4.75 ppm) for weak (ω1/2π ) 30 Hz) continuous-wave (CW) irradiation to saturate the solvent water resonance for the remainder of the d1 period. During the rest of the experiment, the 1H carrier is centered in the middle of the methyl region (0.75 ppm), and proton pulses are applied with a ﬁeld strength of ω1/2π ) 36.8 kHz. Rectangular 13C pulses are centered at 20 ppm and applied with ω1/2π ) 21.4 kHz. The shaped carbon pulses (open domes) have a REBURP proﬁle80 applied on resonance, with a peak RF ﬁeld strength of ω1/2π ) 6.6 kHz and a pulse length of 1.0 ms (at 600 MHz), and thereby refocus only the methyl resonances (bandwidth ∼24 ppm). The other shaped carbon pulses (ﬁlled domes) have a REBURP proﬁle (ω1/2π ) 19.0 kHz, 350 µ s duration at 600 MHz) and are frequency shifted to 40 ppm, so as to cover all aliphatic carbon spins (bandwidth ∼67 ppm). The 13C CW ﬁeld (scheme B) is centered at 25 ppm and applied with a varying strength, satisfying the relationship νCW ) 2 · k · νCPMG (where k is an integer number). Proton and deuterium (78) Hartel, A. J.; Lankhorst, P. P.; Altona, C. Eur. J. Biochem. 1982, 129, 343–357. (79) Orbons, L. P.; van der Marel, G. A.; van Boom, J. H.; Altona, C. Eur. J. Biochem. 1987, 170, 225–239. (80) Geen, H.; Freeman, R. J. Magn. Reson. 1991, 93, 93–141. 17008 J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 Otten et al. decoupling are achieved by WALTZ-16,81 using ω1/2π ) 7.0 and 0.7 kHz and the carrier positions at 0.75 and 4.75 ppm, respectively. Flanking 90° deuterium pulses are given at the same ﬁeld strength and frequency. Values of the delays are τ ) 1.98 ms, TCP ) constant-time CPMG relaxation period, τc ) variable timing in the CPMG element, CT ) 28.0 ms, ε ) 0.4 ms, and d1 ) 1.5 s. The gradient strengths in G/cm (length in ms) are g1 ) 5.0 (0.5), g2 ) 3.0 (1.0), g3 ) 15.0 (1.0) and g4 ) 6.0 (0.3), g5 ) -27.15 (1.0), g6 ) 27.0 (0.25). All gradients are applied along the z-axis. The phase cycling is φ1 ) [x,-x], φ2 ) [x,x,-x,-x], φ3 ) [x], φ4 ) [y,-y], and φrec ) [x,-x,x,-x]. Quadrature detection is obtained using the gradient sensitivity enhancement scheme:82 the echo and antiecho signals are collected separately by inverting the sign of g5 together with inversion of φ3. The pulse sequences and parameter ﬁles can be obtained from the authors upon request. Data Processing and Analysis. The data sets were processed with the NMRPipe/NMRDraw software package,83 and peak intensities were extracted using the program Sparky.84 The values of R2, eff were calculated for each νCPMG value using the relationship: R2,eff(νCPMG) ) -1 TCP ln I(νCPMG) I0 where I(νCPMG) and I0 are the intensities of the cross-peaks with and without the CPMG block, respectively, and TCP is the constant- time CPMG relaxation period. The data analysis and error estimation in the experimental data and ﬁtted exchange parameters were performed as described earlier by Mulder et al.28 Brieﬂy, the uncertainty in the ratio of cross- peaks I(νCPMG)/I0 was estimated from the rmsd in the intensities of the duplicate measurements according to the deﬁnition of pooled relative standard deviation, σr ) (∑(∆ I/Iavg)2/2N)1/2.28,85 The exchange parameters can be obtained from ﬁtting the relaxation dispersion proﬁles (R2, eff as a function of νCPMG) to approximate analytical expressions for chemical exchange (see below) or by numerically solving the Bloch-McConnell equa- tions.86 The dispersion proﬁles for NtrCr obtained at two B0 ﬁelds were ﬁtted together to three different models assuming (i) no exchange, (ii) a two-site fast-exchange process,87 and (iii) a general two-site exchange process (described by Carver and Richardson and Jen, CRJ equation;88,89 see eq 3 of Millet et al.90). In model 1, the experimental data points are ﬁtted to ﬂat lines with R2, eff (νCPMGf∞) at each B0 ﬁeld as adjustable parameters. In the second model, there are two additional parameters, Φex ) pApBδω2 and τex, included to describe the experimental data. In the case where the exchange is not fast (model 3, CRJ equation), it is often possible to separate the product Φex into its components, and the ﬁve ﬁtting parameters in this case are R2, eff (νCPMGf∞) at the two B0 ﬁelds, pA, δω, and τex. The increasing complexity of the functions will generally improve the ﬁt, and, therefore, F-statistics were used to determine whether the description by a more complicated model was justiﬁed at the 99.9% conﬁdence interval. The uncertainties in (81) Shaka, A. J.; Keeler, J.; Frenkiel, T.; Freeman, R. J. Magn. Reson. 1983, 52, 335–338. (82) Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114, 10663–10665. (83) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277–293. (84) Goddard, T. D.; Kneller, D. G. Sparky 3; University of California: San Francisco, CA, 2003. (85) Demers, J. P.; Mittermaier, A. J. Am. Chem. Soc. 2009, 131, 4355– 4367. (86) McConnell, H. M. J. Chem. Phys. 1958, 28, 430–431. (87) Luz, Z.; Meiboom, S. J. Chem. Phys. 1963, 39, 366–370. (88) Carver, J. P.; Richards, R. E. J. Magn. Reson. 1972, 6, 89–105. (89) Jen, J. J. Magn. Reson. 1978, 30, 111–128. (90) Millet, O.; Loria, J. P.; Kroenke, C. D.; Pons, M.; Palmer, A. G. J. Am. Chem. Soc. 2000, 122, 2867–2877.