Joural of the American Chemical Society Article keep the possib eA39GF es 1.65 n fron te.in ke with ectations in the xchang ond the the ICEST profiles CES ST (A ed c the e Figure 7D shows the as ofiles from er.It is clear and the e s 4 residues l.Thes nay in part a o the fact to the e pa and147+002 s for the the A39G to e he CpMG of192 33s and 14 02 e 8B) nalysis CEST ar CPMG data to ange reactior w o he from a gobal fit CONCLUDING REMARKS synthetic data sets for a pair ofkvalues,20 and 200swith ver the past decade,a significar umber of NMR n P=1.5%and a static magnetic field strength of 11.7 T.To excited protein states h been exch k=200s nd the exch with upper und on the order o5000 syste fast 15 r have,in g e the 0.5 d es an extre 120 240 cited ant for the k.=20 also fac systems ote 0.5 it is i 00 100 tra is the igure 8.Ce CEST th of the ins ir the elds sho CEST sim hen sin t =20 08 ms with k values of ~50 and ~150s 815 d dolerg/10.1021/)30014191 Am.Chem.Soc.2012.134.8148-816exchange model as described previously.18 The exchange parameters, (kex, pE) = (101 ± 18 s−1 , 0.8 ± 0.1%), do not agree with the values (51.6 ± 1 s−1 , 1.65 ± 0.02%) extracted from the CEST fits, although the value of pEkex is reasonably accurate, in keeping with expectations in the slow-exchange limit. Figure 7C plots the |Δω̃| values extracted from separate analyses of CPMG (|Δω̃ CPMG|) and CEST (|Δω̃ CEST|) data, and Figure 7D shows the difference ||Δω̃ CPMG| − |Δω̃ CEST|| as a function of residue number. It is clear that there are substantial deviations [pairwise root-mean-square deviation (rmsd) of 2.4 ppm]. These differences may in part arise from the fact that the dispersion profiles are sensitive to the third state that was inferred from CEST data on the basis of the substantial Rex E values for some of the backbone 15N nuclei of the A39G FF domain, although this is hard to establish given the small size of the CPMG profiles. It may be possible to perform a combined analysis of the CEST and CPMG data to obtain a more complete description of the exchange reaction, although we have not done so here. Range of Time Scales That Can Be Characterized. To evaluate the utility of the experiment more fully, we generated synthetic data sets for a pair of kex values, 20 and 200 s−1 , with pE = 1.5% and a static magnetic field strength of 11.7 T. To keep the simulations as realistic as possible, we used 37 “residues” corresponding to those fit from the A39G FF domain with parameters {Δω̃ GE, R1 G, R2 G, R2 E } taken from the experimental fits; these residues showed clear evidence of a second state in the experimental CEST profiles. CEST data were simulated for four ν1 fields (see the Figure 8 caption), and a Gaussian error based on the experimental error was randomly added to each calculated intensity point. We fit each residue independently using profiles from all four ν1 fields, and the distribution of (kex, pE) points obtained for the 24 residues with |Δω̃ GE| > 2.5 ppm is shown in Figure 8. When kex was set to 200 s−1 , extremely accurate exchange parameters were obtained (Figure 8A), with kex and pE values (mean ± standard deviation) of 195 ± 7 s−1 and 1.47 ± 0.02%, respectively. A global fit including all 37 residues yielded kex = 192.5 ± 1.5 s−1 and pE = 1.46 ± 0.01%. The (kex, pE) values were well-defined for kex = 20 s−1 as well, with fitted values of 19.2 ± 3.3 s−1 and 1.4 ± 0.2% (Figure 8B). The values kex = 18.2 ± 0.5 s−1 and pE = 1.48 ± 0.03% were obtained from a global fit including all 37 residues. ■ CONCLUDING REMARKS Over the past decade, a significant number of NMR methods for studying excited protein states have been developed. These include CPMG relaxation dispersion45 and D-evolution12 approaches for characterizing exchanging systems in the approximate regime 200 s−1 ≤ kex ≤ 2000 s−1 ; R1ρ experiments,66 which extend the exchange time scale to higher rates with upper bounds on the order of 50 000 s−1 ; and paramagnetic relaxation enhancement measurements, which are most powerful for systems in the fast-exchange regime.7 Notably, excited states with interconversion rates on the order of ∼200 s−1 or lower have, in general, remained elusive because the slower exchange translates into a smaller effect on the observable ground state. We have shown here that the CEST experiment provides an extremely sensitive and robust approach for obtaining exchange parameters and line widths in the excited state and, most important for the structural work in which we are interested, for measuring accurate excited-state chemical shift values precisely in the slow-exchange limit of interest. Central to the utility of the method is that the experiment, unlike dispersion-based approaches, relies on longitudinal rather than transverse relaxation, with the former often being more than an order of magnitude slower. This enables longer mixing periods to be employed, leading to a “buildup” of the effect of exchange, even in cases where the exchange rates are low. It also facilitates applications to larger protein systems, since for macromolecules such as proteins, T1 values increase as a function of molecular weight. Although T2 values decrease for larger systems, it is important to realize that the major contributor to the line width of the dips in the CEST spectra is the B1 field strength, with the effective line width increasing with applied field. It is possible to decrease the contributions from the intrinsic line width by implementing a TROSY52 version of the experiment. It is worth noting in this regard that relatively large R2 E values were observed for many of the 15N spins in the FF domain excited state (R2 E > 50 s−1 ), yet accurate values of Δω̃could be obtained. Studies at higher static magnetic fields should also be advantageous, since Δω values increase linearly while T1 values increase quadratically with the field in applications involving large molecules. The utility of the CEST experiment has been demonstrated for two experimental systems with kex values of ∼50 and ∼150 s−1 Figure 8. Computations establishing the utility of CEST for studies of slow chemical exchange. Synthetic data were generated as described in the text and fitted as per the experimental data. (A) (kex, pE) distribution from fits of CEST profiles generated with {Δω̃ GE, R1 G, R2 G, R2 E } values taken from the experimental fits of 37 residues for the A39G FF domain that showed distinct dips for the ground and excited states. Values of kex = 200 s−1 and pE = 1.5% were assumed, and the CEST data were simulated (and then simultaneously fit) for four different B1 fields corresponding to frequencies of 50, 25, 12.5, and 6.25 Hz with TEX = 0.4 s. (B) As in (A) but with kex = 20 s−1 and data simulated for 25, 16, 11, and 6 Hz with TEX = 0.8s. Journal of the American Chemical Society Article 8157 dx.doi.org/10.1021/ja3001419 | J. Am. Chem. Soc. 2012, 134, 8148−8161