Microsecond Time Scale Dynamics in Proteins Table 1. Relaxation Dispersion Parameters for Methyl Groups in NtrCr at 25 °Ca,b R2,eff (νCPMG f ∞)d,e (s-1) Rexd,e (s-1) methyl group V9-γ1 V18-γ1 L19-δ2 L40-δ1 L43-δ1 L43-δ2 V50-γ2 I55-γ2 L66-δ2g L76-δ1 L76-δ2 V78-γ1 V78-γ2 I79-γ2 I80-γ2 A83- L87-δ1g A90- V91-γ1 A93- L102-δ2 L114-δ1 11.67 ( 0.22 11.31 ( 0.17 5.85 ( 0.35 5.95 ( 0.12 7.41 ( 0.29 7.44 ( 0.14 -0.02 ( 315.32 5.90 ( 0.11 7.67 ( 0.26 7.51 ( 0.14 7.34 ( 0.41 7.33 ( 0.20 7.66 ( 0.25 7.60 ( 0.13 10.71 ( 1.42 13.97 ( 0.27 7.07 ( 0.21 6.97 ( 0.12 5.87 ( 0.23 5.79 ( 0.15 6.67 ( 0.16 6.59 ( 0.14 8.03 ( 0.15 7.96 ( 0.08 8.68 ( 0.45 8.81 ( 0.14 9.36 ( 0.25 9.10 ( 0.19 9.55 ( 0.32 9.63 ( 0.17 9.32 ( 0.34 9.48 ( 0.15 4.30 ( 1.36 5.37 ( 0.11 9.33 ( 0.75 10.15 ( 0.21 6.78 ( 0.32 7.11 ( 0.08 8.24 ( 0.73 8.62 ( 0.12 0.89 ( 485.4 7.11 ( 0.10 4.14 ( 0.64 4.80 ( 0.10 11.44 ( 0.32 10.80 ( 0.18 5.24 ( 0.62 5.42 ( 0.18 7.01 ( 0.52 7.05 ( 0.24 -6.00 ( 560.80 4.55 ( 0.18 7.01 ( 0.45 6.73 ( 0.23 7.83 ( 0.72 7.81 ( 0.35 5.95 ( 0.45 5.85 ( 0.20 8.36 ( 2.52 14.19 ( 0.41 6.85 ( 0.36 6.67 ( 0.18 5.18 ( 0.43 5.04 ( 0.28 4.69 ( 0.24 4.54 ( 0.19 6.96 ( 0.28 6.83 ( 0.16 8.06 ( 0.79 8.30 ( 0.22 8.65 ( 0.44 8.18 ( 0.31 9.10 ( 0.54 9.25 ( 0.23 8.98 ( 0.56 9.28 ( 0.17 3.07 ( 2.42 4.98 ( 0.17 15.33 ( 1.39 16.79 ( 0.50 6.35 ( 0.57 6.93 ( 0.15 7.93 ( 1.29 8.59 ( 0.17 -5.20 ( 863.3 5.86 ( 0.17 1.99 ( 1.14 3.16 ( 0.17 5.69 5.92 0.72 0.63 1.38 1.36 6.46 0.55 4.50 4.60 8.45 8.46 2.55 2.59 11.78 8.95 2.72 2.79 0.93 0.98 0.62 0.68 1.08 1.13 0.96 0.84 2.08 2.24 3.78 3.73 1.74 1.60 2.49 1.51 6.76 6.11 2.72 2.46 1.18 0.84 6.76 0.56 2.65 2.10 10.13 10.53 1.28 1.12 2.45 2.42 11.49 0.99 8.00 8.18 15.02 15.04 4.53 4.61 20.95 15.92 4.84 4.96 1.65 1.75 1.11 1.20 1.93 2.02 1.70 1.50 3.70 3.98 6.73 6.63 3.09 2.85 4.43 2.68 12.02 10.87 4.84 4.38 2.09 1.49 12.03 0.99 4.72 3.73 A R T I C L E S r2edd,f 1.48 1.51 0.86 0.85 0.89 0.88 0.99 1.02 1.14 1.14 1.21 1.20 1.36 1.34 2.52 2.68 0.96 0.95 0.88 0.87 0.86 0.86 1.07 1.06 0.93 0.92 0.75 0.76 1.23 1.22 1.38 1.37 0.81 0.85 1.25 1.26 1.03 1.04 1.59 1.58 0.94 0.98 1.20 1.23 ∆ δc,d (ppm) 0.209 ( 0.010 0.231 ( 0.003 0.094 ( 0.057 0.076 ( 0.009 0.114 ( 0.033 0.111 ( 0.008 0.971 ( 47.384 0.071 ( 0.009 0.193 ( 0.016 0.204 ( 0.004 0.276 ( 0.019 0.277 ( 0.005 0.148 ( 0.021 0.153 ( 0.005 0.437 ( 0.062 0.285 ( 0.006 0.150 ( 0.016 0.159 ( 0.004 0.083 ( 0.030 0.094 ( 0.010 0.064 ( 0.020 0.078 ( 0.009 0.092 ( 0.019 0.101 ( 0.005 0.108 ( 0.063 0.087 ( 0.009 0.116 ( 0.021 0.142 ( 0.008 0.190 ( 0.021 0.184 ( 0.005 0.139 ( 0.033 0.120 ( 0.005 0.232 ( 0.138 0.117 ( 0.005 0.283 ( 0.041 0.235 ( 0.006 0.179 ( 0.028 0.149 ( 0.004 0.141 ( 0.100 0.087 ( 0.007 1.010 ( 72.520 0.071 ( 0.008 0.202 ( 0.059 0.138 ( 0.005 τex (µ s) 76.3 ( 5.7 64.7 ( 1.9 47.7 ( 37.7 64.7 ( 1.9 62.3 ( 25.6 64.7 ( 1.9 4.0 ( 195.6 64.7 ( 1.9 70.8 ( 9.4 64.7 ( 1.9 65.0 ( 7.1 64.7 ( 1.9 68.3 ( 15.1 64.7 ( 1.9 36.1 ( 6.2 64.7 ( 1.9 71.1 ( 12.1 64.7 ( 1.9 79.2 ( 43.4 64.7 ( 1.9 89.9 ( 42.8 64.7 ( 1.9 75.7 ( 23.9 64.7 ( 1.9 48.3 ( 37.0 64.7 ( 1.9 90.6 ( 26.8 64.7 ( 1.9 61.6 ( 10.1 64.7 ( 1.9 52.7 ( 17.2 64.7 ( 1.9 27.1 ( 17.8 64.7 ( 1.9 49.4 ( 9.9 64.7 ( 1.9 49.7 ( 10.6 64.7 ( 1.9 34.5 ( 28.7 64.7 ( 1.9 3.9 ( 278.3 64.7 ( 1.9 38.1 ( 14.0 64.7 ( 1.9 a Exchange parameters are obtained by ﬁtting the dispersion proﬁles measured at 600 and 800 MHz simultaneously using pB ) 14%. b Methyl groups are stereospeciﬁcally assigned. c ∆ δ was calculated assuming the above-mentioned values for the populations. For other populations (pC and pD), the chemical shift difference can be calculated according to ∆ δCD ) ∆ δAB((pApB)/(pCpD))1/2. d The numbers in the second row indicate values obtained after ﬁtting the proﬁles again, using kex ) 15 458 s-1 as determined from the global ﬁt. e First column ω0/2π ) 600 MHz, and second column ω0/2π ) 800 MHz. f The reduced 2 is calculated using r2ed ) 2/(N - m), where N is the number of experimental data points and m is the number of ﬁtting parameters. g Resonances of δ1/δ2 methyl group overlap, assuming that only the listed probe exhibits an exchange contribution. The dispersion proﬁles of all residues showing an exchange contribution are now accurately described by the fast-exchange model, as evidenced by the reduced 2, and the extracted exchange parameters are given in Table 1 (ﬁrst row for every entry). Only for I55-γ2 the value for r2ed increased signiﬁcantly to 2.5 from an already rather high value of 1.6 in the free ﬁtting procedure. The origin of the poor ﬁt for this residue is not understood at this stage, and it is, therefore, excluded from further analysis. The values obtained for the exchange lifetime are rather similar (see Table 1, ﬁrst row for each probe), suggesting an exchange process between two conformations via a single, cooperative process. A global analysis of the data for all methyl groups in Table 1 (except I55-γ2) was performed, and a value of 64.7 ( 1.9 µ s was obtained for the exchange lifetime. The average red 2 did not change when this global exchange lifetime was used to describe the dispersion proﬁles and thus supports the hypothesis of a single, cooperative exchange process. The results of the global analysis of all CPMG data, using pB ) 14% and τex ) 64.7 µ s, are given in Table 1 (second row for every entry). The exchange rate obtained from the 1H CPMG data, kex ) 15 458 ( 452 s-1, is in excellent agreement with the value of 13 580 ( 850 s-1 obtained earlier from a suite of 15N relaxation data.75 Notwithstanding the agreement between the exchange parameters obtained from the 15N and 1H CPMG relaxation dispersion data, there is an obvious beneﬁt of using the latter approach. In practice, due to the limited attainable 15N CPMG ﬁeld strength, it is only possible to map about one- sixth of the chemical exchange spectral density function, when compared to 1H CPMG relaxation dispersion NMR spectros- copy. Therefore, in previous studies,74,75 it was necessary to extract the exchange parameters from ﬁtting the initial part of the 15N CPMG dispersion proﬁle, and the value of the exchange free component (i.e., R2, 0), obtained from a separate experiment. On the contrary, complete microsecond time scale dynamics dispersion proﬁles are now obtained from a single experiment using the 1H CPMG relaxation experiment (Figure 6). The chemical shift differences between the two exchanging states, determined from the group ﬁt with pB ) 14%, are visualized on the three-dimensional structure of NtrCr in Figure 7. Indeed, all methyl groups identiﬁed to experience a global dynamic J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010 17013