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《生物大分子波谱学原理》课程教学资源（文献资料）Studying “Invisible” Excited Protein States in Slow Exchange with a Major State Conformation

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Studying “Invisible” Excited Protein States in Slow Exchange with a Major State Conformation Pramodh Vallurupalli,*,†,§ Guillaume Bouvignies,†,§ and Lewis E. Kay*,†,‡ † Departments of Molecular Genetics, Biochemistry, and Chemistry, The University of Toronto, Toronto, Ontario, Canada M5S 1A8 ‡ Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 *S Supporting Information ABSTRACT: Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and characterize “invisible” excited protein states in slow exchange with a visible ground-state conformation (excited-state lifetimes ranging from ∼5 to 50 ms) is presented. This method, which is an adaptation of the chemical exchange saturation transfer (CEST) magnetic resonance imaging experiment, involves irradiating various regions of the spectrum with a weak B1 field while monitoring the effect on the visible major-state peaks. The variation in major-state peak intensities as a function of frequency offset and B1 field strength is quantified to obtain the minor-state population, its lifetime, and excited-state chemical shifts and line widths. The methodology was validated with 15N CEST experiments recorded on an SH3 domain−ligand exchanging system and subsequently used to study the folding transition of the A39G FF domain, where the invisible unfolded state has a lifetime of ∼20 ms. Far more accurate exchange parameters and chemical shifts were obtained than via analysis of Carr−Purcell−Meiboom−Gill relaxation dispersion data. ■ INTRODUCTION Proteins are dynamic molecules that are best described in terms of ensembles of interconverting conformations.1,2 The lifetimes of the exchanging conformers, their populations, and indeed their functions can vary significantly within the ensemble.1−4 A quantitative understanding of this important class of biomolecule is therefore predicated on a detailed characterization of the kinetics and thermodynamics of the exchange process- (es)2,5,6 and on the determination of atomic-resolution structures of the many different conformers that populate the proteins’ energy landscape.7−11 This is, in general, difficult to accomplish. Standard methods of structural biology are most successful when applied to a “pure” sample consisting of only a single highly populated conformation. Conformers that are transiently formed and populated at only very low levels, designated in what follows as “invisible” or excited conformational states (excited states for short), have remained recalcitrant to detailed quantitative analysis. This situation is changing, however, with the development of new biophysical approaches, including solution-based NMR methods that “study” exchange processes by monitoring the positions12 and line widths of peaks derived from the “visible” ground state.13−15 Of particular note is the so-called Carr−Purcell− Meiboom−Gill (CPMG) relaxation dispersion experiment,16,17 in which a series of refocusing pulses are applied to the evolving transverse magnetization, leading to a modulation of the chemical shift difference between nuclei in the different exchanging states, |Δω̃| (in ppm), and hence of the effective transverse relaxation of the observed major-state spins.13,14 This modulation can be “fit” to extract the kinetics and thermodynamics of the exchange process as well as the |Δω̃| values for each exchanging spin18 so long as the rates of exchange lie between ∼200 and 2000 s−1 and the fractional population of the excited state is in excess of 0.5%. Over the past decade, CPMG experiments have been extended to studies of protein chemical exchange, from which it has become possible to extract excited-state 1 H, 13C, and 15N backbone chemical shifts19−23 and many of the side-chain chemical shifts24−27 along with the orientations of bond vectors.28,29 Recently, data of this sort has been used to produce atomicresolution models of excited states for a number of different exchanging systems.10,11,30 A significant limitation of the CPMG experiment is the relatively small exchange time scale window over which the interconversion process can be quantified (see above). To address this, we recently introduced an approach whereby CPMG relaxation dispersion profiles are fit in concert with major-state peak shifts in HSQC/HMQC spectra31 that extends the CPMG method to include systems with exchange Received: January 5, 2012 Published: May 3, 2012 Article pubs.acs.org/JACS © 2012 American Chemical Society 8148 dx.doi.org/10.1021/ja3001419 | J. Am. Chem. Soc. 2012, 134, 8148−8161

rates as high as 6000 s−1 . At the opposite end of the exchange spectrum, systems with interconversion rates less than ∼100 s−1 can be studied using magnetization-exchange-based experiments so long as the interconverting states can be observed in spectra.14,32 Cases where the populations are highly skewed (so only peaks from the ground-state conformer are obtained) and the exchange is slow have remained enigmatic, however, because they are not in general amenable to study using either CPMG or typical exchange-type experiments of the sort involving exchange of unperturbed longitudinal magnetization. It is the study of exchanging systems in this regime that we address here. Like the CPMG method, which was developed over 50 years ago16,17 and has subsequently found applications in biomolecular NMR spectroscopy,21,33 the “saturation transfer” class of experiments was originated in the early 1960s by Forsen and Hoffman.34 In these experiments, a weak field is applied at the position of an exchanging peak of interest, and the “perturbation” that results (not necessarily saturation) is transferred to the interconverting state via chemical exchange.35−39 Gupta and Redfield40 applied this approach to study electron exchange between ferri- and ferrocytochrome c in a sample with approximately equal concentrations of reduced and oxidized protein and to assign methyl resonances in the ferro state by saturation transfer from the corresponding wellresolved, hyperfine-shifted peaks of the ferri conformer. Feeney and Roberts used this methodology to assign the chemical shifts of small molecules such as cofactors and inhibitors bound to enzymes. In one such study involving dihydrofolate reductase, 1 H saturation from 2 to 7 ppm in small steps allowed the identification of the small-molecule boundstate peak positions by perturbations to peaks derived from the well-resolved free state.41 Later on, Balaban and colleagues37 and subsequently van Zijl and Yadav42 developed onedimensional chemical exchange saturation transfer (CEST) spectroscopy, by which very low amplitude invisible signals from metabolites and proteins can be amplified manyfold and “read out” from the water resonance so long as the nuclei of interest exchange with water. Clore, Torchia, and co-workers introduced an elegant two-dimensional (2D) experiment, darkstate exchange saturation transfer (DEST), to determine both the kinetics of interconversion between free amyloid-β (Aβ) peptide and very high molecular weight Aβ protofibrils and the 15N line widths of the invisible (“dark”)-state resonances.35 Building on the DEST experiment, we show here that CESTtype experiments can be used to quantify slow chemical exchange precisely in the regime that challenges the CPMG approach, providing the kinetics and thermodynamics of the exchange process as well as the chemical shifts of the excitedstate nuclei often from direct inspection of the resulting spectra. The methodology was cross-validated with a protein−ligand two-state exchanging system that has been studied previously,28 with an exchange rate and minor state population of ∼150 s−1 and 2.5% (5 °C), respectively. Subsequently, the utility of the experiment was demonstrated with an application to the folding of the A39G FF domain,11,43 for which the CPMG dispersion profiles are very small and hence difficult to analyze. Accurate exchange parameters (51.6 ± 1 s−1 and 1.65 ± 0.02%) as well as excited-state chemical shifts were obtained via the CEST methodology. In contrast to other methods for studying invisible states (CPMG or R1,ρ relaxation dispersion, D-evolution), robust exchange parameters were obtained from fits of data recorded on a per-residue basis measured at a single magnetic field strength, allowing a rigorous evaluation of the two-state assumption that is often used in fits of dispersion data. ■ RESULTS AND DISCUSSION Comparison of CPMG and CEST for Studies of Chemical Exchange. Prior to a discussion of experimental details and applications to exchanging systems, we provide a brief discussion of the basic features of the CEST experiment and present an analogy with the CPMG methodology that illustrates some of the similarities between the two approaches. For simplicity, in what follows we consider an isolated 15N spin in a protein exchanging between two conformations, G (ground) and E (excited), G X Y ooo E k k EG GE with distinct chemical shifts ω̃ G and ω̃ E (Δω̃ = ω̃ E − ω̃ G) in each of the two states (Figure 1A). The fractional populations of states G and E, pG ≫ pE, are given by pG = kEG/kex and pE = kGE/kex = 1 − pG, where kex = kGE + kEG. In the cases of interest here, only major-state peaks are visible in the spectra, although in Figure 1A the excited-state correlation (red) is also shown for clarity. To simplify the discussion, we will assume that chemical exchange occurs in the slow-exchange limit, where kex ≪ Δω (i.e., we do not consider multiple transfers of magnetization between the states). Figure 1B illustrates the basic CPMG pulse train that is used to study exchanging systems in the approximate regime 200 s−1 ≤ kex ≤ 2000 s−1 and pE ≥ 0.5%. Here the effective transverse relaxation rates (R2,eff) of the visible peaks are quantified as a function of the frequency νCPMG = 1/(4τCPMG) at which refocusing 180° pulses are applied during a relaxation delay Trelax. The evolution of magnetization is illustrated in Figure 1C, where without any loss in generality it has been assumed that the carrier is placed on resonance for the majorstate correlation being considered (i.e., at ω̃ G). After the 90° pulse at the start of the echo train, magnetization from this state remains aligned along the y axis in the rotating frame while the magnetization from the minor state precesses in the xy plane, losing phase with the ground state. On average, the excitedstate spins accumulate a phase of ⟨ϕ⟩ = Δω/kEG between exchange events, so exchange leads to a reduction in the majorstate signal and hence a nonzero exchange-induced relaxation rate Rex. Each 180° pulse of the CPMG train inverts the sense in which the spins precess around the z axis, reducing the phase accumulation ⟨ϕ⟩ and decreasing Rex. Exchange parameters and minor-state chemical shifts can be obtained from fits of the R2,eff(νCPMG) profile so long as the exchange contribution to R2,eff (i.e., Rex) is quenched as νCPMG increases (Figure 1D). In the slow-exchange limit, Rex = kGE when νCPMG = 0, so the maximum observable change in the R2,eff(νCPMG) profile is kGE. By means of example, consider an exchanging system in the window of interest here with kex = 50 s−1 and pE = 1.5%, for which kGE = 0.75 s−1 . If a contribution of 10 s−1 to R2,eff from intrinsic relaxation is assumed, the maximum loss in the detected signal intensity during a “typical” Trelax delay of 50 ms from chemical exchange, which occurs when νCPMG → 0, is 1 − e−(10.75)(0.05)/e−(10)(0.05) ≈ 3.7%. Quantifying such a small change accurately in the presence of noise and systematic errors is very difficult, compromising the accuracy of the exchange parameters obtained from CPMG relaxation dispersion data in this limit. Journal of the American Chemical Society Article 8149 dx.doi.org/10.1021/ja3001419 | J. Am. Chem. Soc. 2012, 134, 8148−8161

The situation changes, however, when the value of Trelax can be increased significantly, allowing a larger number of exchange events to occur. This can be accomplished by doing experiments that exploit coherences with smaller intrinsic relaxation rates.44−46 For example, when the intrinsic relaxation rate is 1 s−1 and Trelax = 500 ms, the fractional loss of signal due to exchange becomes 1 − e−(1.75)(0.5)/e−(1)(0.5) ≈ 31%, an amount that can easily be quantified. One approach is to use a longitudinal-magnetization-based experiment, since in protein applications the longitudinal relaxation rates (R1) can be an Figure 1. Comparison of the standard CPMG experiment with the weak B1 CEST experiment proposed here. (A) 15N spectrum of an isolated spin exchanging between two states with chemical shifts ω̃ G and ω̃ E. The minor state is shown in the spectrum for purposes of illustration but cannot be observed in the systems of interest. (B) Basic CPMG experiment, with narrow and wide pulses denoting 90° and 180° flip angles, respectively. A variable number (N) of 180° refocusing pulses is applied during a constant-time relaxation element of duration Trelax. (C) Illustration of the mechanism underlying the CPMG experiment, with the major-state peak (blue) assumed to be on resonance. The 180° pulses invert the sense in which “excited-state” spins (red magnetization) precess around the external magnetic field (B0). Stochastic modulation of the chemical shifts of the interconverting spins leads to a dephasing of the magnetization. (D) Typical relaxation dispersion curve obtained by quantifying the peak intensities in a CPMG experiment. (E) Schematic of the CEST experiment. A weak B1 field is applied along the y axis (green) for a time TEX before acquisition of the spectrum. (F) When the B1 field is on resonance with the minor state, precession occurs around the y axis, in analogy to what is shown in (C) for the CPMG experiment. (G) Precession leads to a phase accumulation with respect to the magnetization in the major state and a subsequent reduction in the magnetization intensity of the major state from the constant exchange between states. (H) Intensity profile obtained by quantifying the intensity of the visible-state peak as a function of position of the weak B1 irradiation field. The ratio I/I0 is plotted, where I is the intensity after an irradiation period of duration TEX and I0 is the intensity when TEX = 0. There is a loss of intensity when the weak continuous-wave field is resonant with the major and minor states. Journal of the American Chemical Society Article 8150 dx.doi.org/10.1021/ja3001419 | J. Am. Chem. Soc. 2012, 134, 8148−8161

order of magnitude or more lower than the transverse relaxation rates R2. For this reason, CEST-based experiments offer an attractive avenue for the study of slowly exchanging systems. The CEST experiment is illustrated schematically in Figure 1E; in what follows, we initially assume that R1 = R2 = 0 (but see below). A weak B1 field (ν1 = 5−50 Hz for the studies described here) is applied at a specific offset from the majorstate peak for a time TEX, followed by a 90° pulse and recording of the 15N spectrum. Successive experiments “step” the weak field through the entire spectrum, and the intensity of the visible major-state peak is quantified as a function of offset to detect the position of the corresponding minor-state correlation. When the B1 offset is far from either the major- or minor-state correlation, it has no effect on the spins of interest, and the intensity of the major-state peak is unaffected relative to the case where B1 = 0. However, when the field is placed at ω̃ E (green vector in Figure 1F), it induces Rabi oscillations in the nuclei transiently populating the minor state, leading to precession around the y axis in the xz plane in analogy to the precession about the z axis that occurs in a CPMG experiment (Figure 1C). The bulk magnetization vector corresponding to the excited state rotates on average by an angle ⟨θ⟩ = 2πν1/kEG around the y axis between exchange events (Figure 1G), leading to a net reduction in the polarization of the ground state that is detected, leading to the profile illustrated in Figure 1H. Here the intensity I of the major-state correlation (normalized to I0, the intensity when TEX = 0) is plotted as a function of the position of the B1 field. The reduction in the observed magnetization of state G when the B1 field overlaps with the minor-state peak is clearly seen. This phenomenon is directly analogous to chemical-exchange-induced line broadening, with ν1 in CEST corresponding to Δω/2π (Δω = ωE − ωG) in the CPMG experiment. Therefore, in direct analogy with CPMG relaxation dispersion, the intensity of the major-state correlation as a function of TEX in the pG ≫ pE limit can be calculated from the following approximate expression for Rex derived by Millet et al.:47 I( ) exp( ) T I RT EX 0 ex EX = − (1) where = + ( ) πν R ppk 1 k ex G E ex 2 2 ex 1 The above discussion assumed that that R1 = R2 = 0. In general, the situation is more complicated because relaxation occurs during precession of the magnetization about B1 and potentially also saturation. If exchange is neglected, the time dependence of the z component of the magnetization upon application of an on-resonance B1 field is given by ω ω ω = + + + ⎛ ⎝ ⎜ ⎞ ⎠ M T M ⎟ R R R R f T R R () () z EX 0 1 2 1 2 1 2 EX 1 2 1 2 1 2 (2.1) where ρ ρω ρ ρω = + × − | |≥Δ + × − | |<Δ ⎧ ⎨ ⎪ ⎪⎪ ⎩ ⎪ ⎪ ⎪ f T T RT T R T R T RT T R T R ( ) [cos( ) sinc( )] exp( ), [cosh( ) sinhc( )] exp( ), EX EX avg EX EX avg EX 1 EX avg EX EX avg EX 1 (2.2) and Ravg = (R1 + R2)/2, ΔR = (R2 − R1)/2, ρ = |ω1 2 − (ΔR) 2 | 1/2, and ω1 = γB1. From eqs 2.1 and 2.2, it can be seen that magnetization decays to the steady-state value M0R1R2/(ω1 2 + R1R2) with a time constant Ravg. The corresponding solution of the Bloch equations that includes chemical exchange and a weak B1 field corresponding to the CEST experiment is complicated and offers little understanding in its most general form.48,49 However, some insight can be obtained from the expressions that neglect exchange (eqs 2.1 and 2.2). For example, if the lifetime of the excited state, 1/kEG, is such that 1/kEG ≤ TEX and Ravg/kEG > 1, then the excited-state magnetization approaches its steady-state value [i.e., f(TEX) ≈ 0 in eq 2.1 since exp(−Ravg/kEG) is small], which is close to zero (saturation) even for small values of ω1 and typical relaxation rates (e.g., ω1 = 2π × 10 rad/s, R1 ≈ 1 s−1 , R2 = 5−20 s−1 ). In this case, chemical exchange transfers the saturation from state E to G, with the magnetization in the E state subsequently “replenished” by exchange from G to E, leading to a decrease in the magnetization of the ground state. For many exchanging systems it is not the case that Ravg/kEG > 1, at least for some of the spins, in which case the transferred magnetization (E to G) is only partially saturated [i.e., f(TEX) ≠ 0]. CEST Experiment for Studies of Slowly Exchanging, Highly Skewed Protein Systems. Figure 2 shows the gradient-coherence-selected, enhanced-sensitivity-based pulse scheme for quantifying the exchange parameters and excitedstate chemical shifts in slowly exchanging 15N-labeled protein systems. The basic pulse scheme is essentially a modification of the standard experiment used to measure 15N R1 values in amide groups of proteins;50 only the salient features as they pertain to the CEST experiment will be described here. The magnetization transfer pathway is summarized succinctly as → ⎯→⎯ → ⎯→ ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ − ab c d e H() N() N() N( ) H () z z T z x t x y 1 15 15 15 /SE RINEPT 1 / EX 1 (3) Briefly, the amide proton z magnetization at point a is transferred via a refocused INEPT element51 to 15N longitudinal magnetization at point b. The 1 H and 15N carriers, originally on the water 1 H resonance and in the middle of the amide 15N spectrum, respectively, are positioned in the center of the amide 1 H region and at the desired position for weak 15N irradiation during the subsequent TEX period. 1 H composite pulse decoupling is applied during this interval, effectively reducing the 15N−1 H spin system to an isolated 15N spin. At the end of the TEX period (point c), the 1 H and 15N carriers are returned to their original positions, and the 15N transverse magnetization evolves during the subsequent t1 period followed by transfer to 1 H for detection during t2. The intensities of the cross-peaks in the resulting 2D 15N−1 H spectra are quantified to obtain the exchange parameters and excited-state 15N chemical shifts as described later. The basic pulse scheme is similar to that recently used in a study of Aβ peptide protofibril exchange dynamics,35 except that significantly larger 15N B1 “saturating” fields were used there (ν1 ≈ 170 Hz vs 5−50 Hz in the present work) with 15N TROSY/anti-TROSY components52,53 interconverted through the application of 1 H 180° pulses at 100 ms intervals. In cases where excited-state chemical shifts are to be measured, it is preferable to use very weak B1 fields, since the peak line widths increase with B1 (see below). 1 H decoupling is more critical in these cases because ν1 ≪ JHN, where JHN is the Journal of the American Chemical Society Article 8151 dx.doi.org/10.1021/ja3001419 | J. Am. Chem. Soc. 2012, 134, 8148−8161

one-bond 1 H−15N scalar coupling constant, so the 15N B1 field by itself is not sufficient to achieve adequate decoupling. Here we used a simple 1 H composite pulse decoupling scheme that was tested to ensure that decoupling sidebands would not give rise to spurious intensity dips at positions distinct from the major and minor states. Figure 3 shows results from a number of different decoupling sequences that were examined with the scheme in Figure 2 using a concentrated sample of protein L, a small 63 residue protein for which no excited states have been detected in previous work. “Intensity dips” are therefore expected only at peak positions that are measured in a regular HSQC spectrum (Figure 3A). However, standard decoupling sequences such as WALTZ-1659 produce (small) sidebands in the decoupled 15N spectrum, and when the carrier of the 15N B1 field is positioned on one of these sidebands, the major-state peak is modulated, leading to intensity dips at frequencies that do not correspond to protein resonance positions (Figure 3B, arrows). This complicates interpretation of the data. Repetition of a single (composite) 1 H inversion pulse (no supercycling) produces sidebands ±1/ (2pwINV) Hz from the position of the decoupled 15N peak, where pwINV is the length of the inversion pulse. In general, the decoupling “artifacts” are much farther from the major peak than for the WALTZ scheme. In fact, so long as the inversion pulse is short, the first sidebands can be placed far from any of the protein major (minor) peaks, ensuring that “inadvertent” excitation does not occur. We tested a pair of composite inversion pulses, 90x180y270x 59 [Figure 3C, pwINV = 1.5/(1 H field)] and 90x240y90x 54 [Figure 3D, pwINV = 1.17/(1 H field)], both of which have excellent inversion properties over bandwidths sufficiently wide for the applications considered here. Using a 2.35 kHz 1 H field (11.7 T B0 field) produces sidebands ±1/(2pwINV) Hz from the major-state 15N resonance, corresponding to ∼782.5 Hz/15.5 ppm for 90x180y270x and ∼1004 Hz/20.0 ppm for 90x240y90x, leading to very small spurious peaks in some of the CEST traces (Figure 3C,D). Of the two composite pulses considered, we prefer the 90x240y90x pulse because it is shorter and produces side bands further away from protein resonances, most often outside the 15N chemical shift window. Finally, as a gauge of the level of artifacts introduced by 1 H decoupling, it is worth noting that we purposefully chose a test sample with a very high protein concentration (4 mM); these (tiny) artifacts will most certainly not be observed in standard samples with concentrations on the order of 1 mM. Cross-Validation of the Methodology. In an application of CEST that is most similar to the work described here, Clore, Torchia, and co-workers used a 2D 15N-based experiment to quantify the exchange between an Aβ peptide and a multimegadalton protofibril complex.35 Because of the very large size of the protofibril, the 15N transverse relaxation rates in the bound form are extremely large, on the order of 20 000 s−1 , precluding measurement of bound-state chemical shifts. In the applications considered here, we wish to use 2D CEST as a complement to CPMG relaxation dispersion in cases where the dispersion experiment fails, and our goal is to obtain both exchange parameters and excited-state chemical shifts. The latter requirement necessitates the use of weaker B1 fields than in other studies and effective 1 H decoupling schemes that efficiently collapse the 15N multiplet structure, increasing both the resolution and sensitivity, without introducing decoupling sideband artifacts that can complicate interpretation of the data. Therefore, it was important to cross-validate the approach using an exchanging system where the “answer” is already known. Here we focused on a protein (Abp1p SH3 domain)−ligand (Ark1p peptide) exchanging system that we have used previously.28 To a 1 mM sample of 15N-labeled SH3 domain was added a small amount of unlabeled peptide, corresponding to a bound mole fraction of 0.025. Under these conditions at Figure 2. Pulse scheme for the weak B1 CEST experiment for studying slow-time-scale chemical exchange at 15N sites in the backbone amide groups of proteins. 1 H and 15N 90° and 180° pulses are shown as narrow and wide black bars, respectively, and unless indicated otherwise are applied along the x axis at the maximum available power (2pw is the duration of the 1 H 180° pulse). The 1 H transmitter is positioned on the water resonance throughout the sequence except between points b and c, when it is moved to the center of the amide region (8.4 ppm). Similarly, the 15N transmitter is placed at 119 ppm except between points b and c, when it is relocated to the desired offset (a set of 2D spectra are recorded with different offsets). Typical values of the 15N B1 field range between 5 and 55 Hz. A coherent decoupling train consisting of 90x240y90x pulses54 is used for 1 H decoupling between points b and c (∼2.5 and ∼4 kHz for 11.7 and 18.8 T, respectively). The phase cycle is ϕ1 = {x, −x}, ϕ2 = {y}, ϕ3 = {2x, 2y, 2(−x), 2(−y)}, ϕ4 = {x}, receiver = {x, −x, −x, x} (the experiment can be performed with a minimum four-step cycle). Gradient strengths in G/cm (with corresponding lengths in ms given in parentheses) are g1 = 5(1), g2 = 4(0.5), g3 = 10(1), g4 = 8(0.5), g5= 7(0.5), g6 = −25(1), g7 = 15(1.25), g8 = 4(0.5), g9 = 8(0.5), g10 = 29.6(0.125). Weak bipolar gradients g0 = 0.1 G/cm with opposite signs are applied during each half of the t1 period. Quadrature detection is achieved via the enhanced-sensitivity55 gradient method,56,57 whereby separate data sets are acquired for each t1 increment corresponding to (g10, ϕ4) and (−g10, −ϕ4). ϕ2 and the receiver phase are incremented in a States-TPPI manner.58 Delays are set to the following values: τa = 2.25 ms, τb = 2.75 ms, and τc = 0.75 ms. 15N decoupling during acquisition is achieved via WALTZ-16.59 To ensure that heating from 1 H decoupling is independent of the duration of TEX, 1 H decoupling is applied for a time TMAX − TEX immediately after the completion of acquisition, where TMAX is the maximum exchange time used. A recycle delay of 1.5 s is used between scans. A reference experiment, recorded with TEX = 0 s, is included in fits of data so that accurate R1 G values can be obtained. Journal of the American Chemical Society Article 8152 dx.doi.org/10.1021/ja3001419 | J. Am. Chem. Soc. 2012, 134, 8148−8161

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