Microsecond Time Scale Dynamics in Proteins protein folding,6-10 ligand binding,10,11 enzyme catalysis,12-18 and allosteric regulation19 have appeared. Relaxation dispersion NMR spectroscopy investigates the inﬂuence of slow (µ s-ms) stochastic processes on the nuclear spin relaxation. For a molecule undergoing a conformational exchange process described by kAB A y\z B kBA relaxation dispersion experiments yield information on (i) the exchange rate, kex ) 1/τex ) kAB + kBA, (ii) the populations of the exchanging states, pA and pB, and (iii) the absolute difference between the chemical shifts of state A and B, |∆ ω|.20,21 While the basic building block of these experiments, the spin-echo,datesbacktothe1950s,22Carr-Purcell-Meiboom-Gill (CPMG)23,24 relaxation dispersion spectroscopy has emerged over the past decades as a powerful tool to study motions on millisecond to second time scales in proteins, through combined advances in biochemical labeling techniques and NMR pulse scheme developments (see, for example, Lund- stro¨ m et al. and references therein25). In particular, for 13C CPMG experiments, it has been vital to prepare NMR samples with isolated 13C probes to remove artifacts in CPMG data sets due to the large one-bond and nonvanishing long- range homonuclear scalar coupling constants.26-28 As a result, a host of experiments are now available that exploit protein backbone nuclei (15N, 1HN, 1HR, 13CR, and 13C)6,7,29-39 as well (6) Hill, R. B.; Bracken, C.; DeGrado, W. F.; Palmer, A. G. J. Am. Chem. Soc. 2000, 122, 11610–11619. (7) Tollinger, M.; Skrynnikov, N. R.; Mulder, F. A. A.; Forman-Kay, J. D.; Kay, L. E. J. Am. Chem. Soc. 2001, 123, 11341–11352. (8) Korzhnev, D. M.; Salvatella, X.; Vendruscolo, M.; Di Nardo, A. A.; Davidson, A. R.; Dobson, C. M.; Kay, L. E. Nature 2004, 430, 586– 590. (9) Zeeb, M.; Balbach, J. J. Am. Chem. Soc. 2005, 127, 13207–13212. (10) Sugase, K.; Dyson, H. J.; Wright, P. E. Nature 2007, 447, 1021– 1027. (11) Mulder, F. A. A.; Mittermaier, A.; Hon, B.; Dahlquist, F. W.; Kay, L. E. Nat. Struct. 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These experiments have been employed to characterize exchange processes on the millisecond time scale, using maximum pulse repetition rates on the order of 1 kHz. This limitation is primarily a consequence of signiﬁcant heating of the NMR sample at high duty cycles, as a result of dielectric losses, but hardware issues (e.g., power handling of the ampliﬁers and probehead and detuning of the RF circuit) also play a role. The experimental constraints are less stringent if 1H is used as nucleus to record the CPMG relaxation dispersion proﬁles, because sample heating at a certain duty cycle is much lower in that case.45 The ability to use a wide range of effective CPMG ﬁeld strengths makes it possible to accurately address faster processes (down to ∼10 µ s) by sampling the spectral density curve over a wider range. It is also vital for the detection of multiple exchange processes occurring on different time scales. In addition, 1H CPMG experiments have several other advan- tages. 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