New probes of ligand flexibility in drug design: transferred (13)C CSA-dipolar cross-correlated relaxation at natural abundance

Understanding the impact of molecular flexibility remains an important outstanding problem in rational drug design. Toward this end, we present new NMR relaxation methods that describe ligand flexibility at the atomic level. Specifically, we measure natural abundance (13)C cross-correlated relaxation parameters for ligands in rapid exchange between the free and receptor-bound states. The rapid exchange transfers the bound state relaxation parameters to the free state, such that a comparison of relaxation rates in the absence and presence of protein receptor yields site-specific information concerning the bound ligand flexibility. We perform these measurements for aromatic carbons, which are highly prevalent in drug-like molecules and demonstrate significant cross-correlated relaxation between the (13)C-(1)H dipole-dipole (DD) and (13)C chemical shift anisotropy (CSA) relaxation mechanisms. Our use of natural abundance measurements addresses the practical difficulties of obtaining isotope-labeled ligands in pharmaceutical research settings. We demonstrate our methods on a small ligand of the 42 kDa kinase domain of the p38 MAP kinase. We show that exchange-transferred cross-correlated relaxation measurements are not only sensitive probes of bound ligand flexibility but also offer complementary advantages over standard R(1) = 1/T(1) and R(2) = 1/T(2) measurements. The ligand flexibility profiles obtained from the relaxation data can help assess the influence of dynamics on ligand potency or pharmacokinetic properties or both, and thereby include inherent molecular flexibility in drug design.     

Polymer-Solvent Interactions in Solutions of Thermoresponsive Polymers Studied by NMR and IR Spectroscopy

Summary: NMR relaxation and diffusion coefficient measurements revealed that a portion of water molecules is bound in mesoglobules formed in poly(N-isopropylmethacrylamide) (PIPMAm) and poly(vinyl methyl ether) (PVME) aqueous solutions above the LCST, with fast exchange between bound and free states (residence time ∼1 ms). Two types of bound water molecules were assigned to water bound inside mesoglobules and on their surface. For highly concentrated PVME/D₂O solutions (c ≥ 20 wt%) a slow exchange was detected by NMR for bound water (residence time = 2.1 s). For PIPMAm aqueous solution IR spectra indicate a two-steps character of the phase transition. For PIPMAm in D₂O/ethanol (EtOH) mixtures the globular structures were observed by NMR at 298 K for certain compositions of the mixed solvent (cononsolvency effect). Virtually no EtOH is bound in these globular structures, in contrast to the temperature-induced globular structures.     

Mapping allostery through equilibrium perturbation NMR spectroscopy

The understanding of allostery relies on the comparative analysis of macromolecules in their free and bound states. However, the direct free versus bound comparison is often challenging due to the instability of one of the two forms. This problem is effectively circumvented by using minor free/bound equilibrium perturbations which are tolerated without compromising sample stability. The subtle equilibrium perturbations are still able to reveal significant apo/holo differences if monitored by NMR experiments that are sensitive to minor populations within dynamic equilibria, such as NMR relaxation dispersion (NMRD) and hydrogen exchange (H/D and H/H) rates. These measurements are complementary to each other as they unmask how a ligand affects both the stable and the excited states of the free energy landscape for its protein receptor. The proposed equilibrium perturbation approach therefore significantly expands the scope of applicability of NMRD and hydrogen exchange experiments to the investigation of ligand-protein interactions, in general, unveiling allosteric "hot spot" maps for systems that have been traditionally elusive to direct free/bound comparisons.     

Pulsed field gradient NMR study of phenol binding and exchange in dispersions of hollow polyelectrolyte capsules

The distribution and exchange dynamics of phenol molecules in colloidal dispersions of submicron hollow polymeric capsules is investigated by pulsed field gradient NMR (PFG-NMR). The capsules are prepared by layer-by-layer assembly of polyelectrolyte multilayers on silica particles, followed by dissolution of the silica core. In capsule dispersion, (1)H PFG echo decays of phenol are single exponentials, implying fast exchange of phenol between a free site and a capsule-bound site. However, apparent diffusion coefficients extracted from the echo decays depend on the diffusion time, which is typically not the case for the fast exchange limit. We attribute this to a particular regime, where apparent diffusion coefficients are observed, which arise from the signal of free phenol only but are influenced by exchange with molecules bound to the capsule, which exhibit a very fast spin relaxation. Indeed, relaxation rates of phenol are strongly enhanced in the presence of capsules, indicating binding to the capsule wall rather than encapsulation in the interior. We present a quantitative analysis in terms of a combined diffusion-relaxation model, where exchange times can be determined from diffusion and spin relaxation experiments even in this particular regime, where the bound site acts as a relaxation sink. The result of the analysis yields exchange times between free phenol and phenol bound to the capsule wall, which are on the order of 30 ms and thus slower than the diffusion controlled limit. From bound and free fractions an adsorption isotherm of phenol to the capsule wall is extracted. The binding mechanism and the exchange mechanism are discussed. The introduction of the global analysis of diffusion as well as relaxation echo decays presented here is of large relevance for adsorption dynamics in colloidal systems or other systems, where the standard diffusion echo decay analysis is complicated by rapidly relaxing boundary conditions.     

NMR and SANS studies of aggregation and microemulsion formation by phosphorus fluorosurfactants in liquid and supercritical carbon dioxide

(1)H NMR relaxation and diffusion studies were performed on water-in-CO(2) (W/C) microemulsion systems formed with phosphorus fluorosurfactants of bis[2-(F-hexyl)ethyl] phosphate salts (DiF(8)), having different counterions (Na(+), NH(4)(+), N(CH(3))(4)(+)) by means of high-pressure in situ NMR. Water has a low solubility in CO(2) and is mainly solubilized by the microemulsion droplets formed with surfactants added to CO(2) and water mixtures. There is rapid exchange of water between the bulk CO(2) and the microemulsion droplets; however, NMR relaxation measurements show that the entrapped water has restricted motion, and there is little "free" water in the core. Counterions entrapped by the droplets are mostly associated with the surfactant headgroups: diffusion measurements show that counterions and the surfactant molecules move together with a diffusion coefficient that is associated with the droplet. The outer shell of the microemulsion droplets consists of the surfactant tails with some associated CO(2). For W/C microemulsions formed with the phosphate-based surfactant having the ammonia counterion (A-DiF(8)), the (1)H NMR signal for NH(4)(+) shows a much larger diffusion coefficient than that of the surfactant tails. This apparent paradox is explained on the basis of proton exchange between water and the ammonium ion. The observed dependence of the relaxation time (T(2)) on W(0) (mole ratio of water to surfactant in the droplets) for water and NH(4)(+) can also be explained by this exchange model. The average hydrodynamic radius of A-DiF(8) microemulsion droplets estimated from NMR diffusion measurements (25 degrees C, 206 bar, W(0) = 5) was R(h) = 2.0 nm. Assuming the theoretical ratio of R(g)/R(h) = 0.775 for a solid sphere, where R(g) is the radius of gyration, the equivalent hydrodynamic radius from SANS is R(h) = 1.87 nm. The radii measured by the two techniques are in reasonable agreement, as the two techniques are weighted to measure somewhat different parts of the micelle structure.     

Slow exchange in the chromophore of a green fluorescent protein variant

Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.     

Quantitative analysis of conformational exchange contributions to 1H-15N multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant

Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential 1H-15N multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 +/- 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the 1H and 15N chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential 1H-15N multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from 15N off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the 1H and 15N spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the 1H chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on 1H single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.     

Singlet-state exchange NMR spectroscopy for the study of very slow dynamic processes

Singlet states with lifetimes that are longer than spin-lattice relaxation times TS > T1 offer unique opportunities for studying very slow dynamic processes in solution-state NMR. A set of novel experiments can achieve broadband excitation of singlet states in pairs of coupled spins. The most elaborate of these experiments, two-dimensional singlet-state exchange spectroscopy (SS-EXSY), is independent of the offsets of the two spins, their relative chemical shifts, and their scalar couplings. The new methods open the way to study very slow chemical exchange or translational diffusion using mixing times taum = Ts > T1. The lifetimes TS of singlet states of pairs of protons in a partially deuterated saccharide are shown to be longer than the longitudinal proton relaxation times T1 in the same compound by a factor of ca. 37.     

Deuteron Solid-State NMR Relaxation Measurements Reveal Two Distinct Conformational Exchange Processes in the Disordered N-Terminal Domain of Amyloid-β Fibrils

We employed deuterium solid-state NMR techniques under static conditions to discern the details of the μs–ms timescale motions in the flexible N-terminal subdomain of Aβ_1–40 amyloid fibrils, which spans residues 1–16. In particular, we utilized a rotating frame (R_1ρ) and the newly developed time domain quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) relaxation measurements at the selectively deuterated side chains of A2, H6, and G9. The two experiments are complementary in terms of probing somewhat different timescales of motions, governed by the tensor parameters and the sampling window of the magnetization decay curves. The results indicated two mobile “free” states of the N-terminal domain undergoing global diffusive motions, with isotropic diffusion coefficients of 0.7−1 ⋅ 10⁸ and 0.3−3 ⋅ 10⁶ad² s⁻¹. The free states are also involved in the conformational exchange with a single bound state, in which the diffusive motions are quenched, likely due to transient interactions with the structured hydrophobic core. The conformational exchange rate constants are 2−3 ⋅ 10⁵ s⁻¹ and 2−3 ⋅ 10⁴ s⁻¹ for the fast and slow diffusion free states, respectively.     

Slow dynamics in folded and unfolded states of an SH3 domain.

(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.     

Cooperative interaction of n-butylammonium ion with 1,3-alternate tetrapropoxycalix [4]arene: NMR and theoretical study

The interaction of 1,3-alternate tetrapropoxycalix[4]arene (1) with n-butylammonium ion (2) in CD(2)Cl(2) was examined using (1)H, (13)C and (14)N NMR spectroscopy and DFT (density functional theory) calculations. NMR shows that 1 forms with 2 an equimolecular hydrogen-bonded complex with the equilibrium constant 5.91 x 10(3) l/mol at 296 K. The structure of the complex can be shown to be asymmetric at 203 K, with 2 interacting by hydrogen bonds with the two ethereal oxygen atoms of one half of 1 and with the pi system of the other half, but is rapidly averaged to an apparent C(4h) symmetry by chemical exchange at higher temperatures. Using two related but independent techniques based on transverse and rotating-frame proton relaxation, it is shown that only an intermolecular exchange of 2 between the bound and free states takes place, in contrast to previously studied interaction of 1 with H(3)O(+). Its correlation time is 0.169 ms. It is shown by DFT calculations that such swift exchange is not possible without a cooperative interaction of both 2 and 1 with several molecules of water present. Similarities and contrasts between the exchange processes of 2 and H(3)O(+) bound to 1 are discussed, in particular with respect to the apparent quantum tunneling of the latter inside the molecule of the complex.     

An analytical model for probing ion dynamics in clays with broadband dielectric spectroscopy

A simple two-state model is proposed to explicitly derive the ionic contribution to the frequency-dependent dielectric permittivity of clay. This model is based on a separation of time scales and accounts for two possible solvation modes (inner/outer-sphere complexes) for ions in the interlayer spacing and a possible chemical exchange between both forms. The influence on the permittivity of thermodynamic (distribution constant K(d)) and dynamic (diffusion coefficient, chemical relaxation rate) parameters is discussed. In turn, this model is used to analyze experimental data obtained with Na-montmorillonite for two relative humidities. The values of the parameters extracted from these measurements, and their variation with water content, show that the proposed model is at least reasonable.     

Protein binding to lanthanide(III) complexes can reduce the water exchange rate at the lanthanide

The GdIII-based magnetic resonance imaging contrast agent MS-325 targets the blood protein serum albumin, resulting in an increased efficacy (relaxivity) as a relaxation agent. MS-325 showed different relaxivities when bound to serum albumin from different species, e.g., r1=30.5 mM-1 s-1 (rabbit) vs 46.3 mM-1 s-1 (human) at 35 degrees C and 0.47 T. To investigate the mechanism for this difference, surrogate complexes were prepared where the GdIII ion was replaced by other LnIII ions. Fluorescence lifetime measurements of the EuIII analogue indicated that the hydration number was q=1 and did not change when bound to either human, rat, rabbit, pig, or dog serum albumin. The YbIII analogue, YbL1, was prepared and characterized by 1H NMR. Line-shape analysis of the paramagnetic-shifted 1H NMR resonances in the presence of increasing amounts of human (HSA) or rabbit (RSA) serum albumin allowed estimation of the transverse relaxation rate, R2, of these resonances for the protein-bound YbL1. The rotational correlation time of YbL1 was calculated from R2, and the Yb-H distance and was tauR=8+/-1 ns when bound to HSA and 13+/-2 ns when bound to RSA. The water exchange rate at the DyIII analogue, DyL1, was determined from variable-temperature R2 measurements at 9.4 T when DyL1 was bound to either HSA or RSA. At 37 degrees C, water exchange at DyL1 was (31+/-5)x10(6) s-1 when bound to HSA but (3.8+/-0.2)x10(6) s-1 when bound to RSA. Slower water exchange upon RSA binding explains the differences in relaxivity observed. The approach of using surrogate lanthanides to identify specific molecular parameters influencing relaxivity is applicable to other protein-targeted GdIII contrast agents.     

Quantitative characterization of food products by two-dimensional D-T2 and T1-T2 distribution functions in a static gradient

We present new NMR techniques to characterize food products that are based on the measurement of two-dimensional diffusion-T2 relaxation and T1-T2 relaxation distribution functions. These measurements can be performed in magnets of modest strength and low homogeneity and do not require pulsed gradients. As an illustration, we present measurements on a range of dairy products that include milks, yogurt, cream, and cheeses. The two-dimensional distribution functions generally exhibit two distinct components that correspond to the aqueous phase and the liquid fat content. The aqueous phase exhibits a relatively sharp peak, characterized by a large T1/T2 ratio of around 4. The diffusion coefficient and relaxation times are reduced from the values for bulk water by an amount that is sample specific. The fat signal has a similar signature in all samples. It is characterized by a wide T2 distribution and a diffusion coefficient of 10(-11) m2/s for a diffusion time of 40 ms, determined by bounded diffusion in the fat globules of 3 microm diameter.     

Ultrafast two-dimensional infrared vibrational echo chemical exchange experiments and theory

Ultrafast two-dimensional (2D) infrared vibrational echo experiments and theory are used to examine chemical exchange between solute-solvent complexes and the free solute for the solute phenol and three solvent complex partners, p-xylene, benzene, and bromobenzene, in mixed solvents of the partner and CCl4. The experiments measure the time evolution of the 2D spectra of the hydroxyl (OD) stretching mode of the phenol. The time-dependent 2D spectra are analyzed using time-dependent diagrammatic perturbation theory with a model that includes the chemical exchange (formation and dissociation of the complexes), spectral diffusion of both the complex and the free phenol, orientational relaxation of the complexes and free phenol, and the vibrational lifetimes. The detailed calculations are able to reproduce the experimental results and demonstrate that a method employed previously that used a kinetic model for the volumes of the peaks is adequate to extract the exchange kinetics. The current analysis also yields the spectral diffusion (time evolution of the dynamic line widths) and shows that the spectral diffusion is significantly different for phenol complexes and free phenol.     

Dynamics of xenon binding inside the hydrophobic cavity of pseudo-wild-type bacteriophage T4 lysozyme explored through xenon-based NMR spectroscopy

Wild-type bacteriophage T4 lysozyme contains a hydrophobic cavity with binding properties that have been extensively studied by X-ray crystallography and NMR. In the present study, the monitoring of 1H chemical shift variations under xenon pressure enables the determination of the noble gas binding constant (K = 60.2 M(-1)). Although the interaction site is highly localized, dipolar cross-relaxation effects between laser-polarized xenon and nearby protons (SPINOE) are rather poor. This is explained by the high value of the xenon-proton dipolar correlation time (0.8 ns), much longer than the previously reported values for xenon in medium-size proteins. This indicates that xenon is highly localized within the protein cavity, as confirmed by the large chemical shift difference between free and bound xenon. The exploitation of the xenon line width variation vs xenon pressure and protein concentration allows the extraction of the exchange correlation time between free and bound xenon. Comparison to the exchange experienced by protein protons indicates that the exchange between the open and closed conformations of T4 lysozyme is not required for xenon binding.     

Recent developments in the characterization of water interacting with proteins by 17O NMR

Transverse and longitudinal ¹⁷O-water relaxation rates were detected in different samples of BSA solutions after one-quantum and triple-quantum-filtered NMR sequences. Another contribution other than quadrupolar relaxation was found for transverse relaxation, which did not change significantly with the concentration and hence could not correspond to agglomeration of proteins. This was interpreted as chemical exchange between different types of ¹⁷O-water in fast motion; probably free water and water weakly bound to the proteins. At lower BSA concentrations, two peaks were detected for water; this was in agreement with this hypothesis. The interactions between BSA and lactic acid were also studied. It was shown that at a sufficient concentration of lactic acid, the number of strongly bound water molecules detected diminishes. On the other hand, the weakly bound water properties do not change significantly.     

Orientational relaxation dynamics in aqueous ionic solution: polarization-selective two-dimensional infrared study of angular jump-exchange dynamics in aqueous 6M NaClO4

The dynamics of hydrogen bond (H-bond) formation and dissociation depend intimately on the dynamics of water rotation. We have used polarization resolved ultrafast two-dimensional infrared (2DIR) spectroscopy to investigate the rotational dynamics of deuterated hydroxyl groups (OD) in a solution of 6M NaClO(4) dissolved in isotopically mixed water. Aqueous 6M NaClO(4) has two peaks in the OD stretching region, one associated with hydroxyl groups that donate a H-bond to another water molecule (OD(W)) and one associated with hydroxyl groups that donate a H-bond to a perchlorate anion (OD(P)). Two-dimensional IR spectroscopy temporally resolves the equilibrium inter conversion of these spectrally distinct H-bond configurations, while polarization-selective 2DIR allows us to access the orientational motions associated with this chemical exchange. We have developed a general jump-exchange kinetic theory to model angular jumps associated with chemical exchange events. We use this to model polarization-selective 2DIR spectra and pump-probe anisotropy measurements. We determine the H-bond exchange induced jump angle to be 49 ± 5° and the H-bond exchange rate to be 6 ± 1 ps. Additionally, the separation of the 2DIR signal into contributions that have or have not undergone H-bond exchange allows us to directly determine the orientational dynamics of the OD(W) and the OD(P) configurations without contributions from the exchanged population. This proves to be important because the orientational relaxation dynamics of the populations that have undergone a H-bond exchange differ significantly from the populations that remain in one H-bond configuration. We have determined the slow orientational relaxation time constant to be 6.0 ± 1 ps for the OD(W) configuration and 8.3 ± 1 ps for the OD(P) configuration. We conclude from these measurements that the orientational dynamics of hydroxyl groups in distinct H-bond configurations do differ, but not significantly.     

Frequency-dependent dielectric permittivity of salt-free charged lamellar systems

We present a new model to analyze dielectric spectroscopy measurements on charged lamellar systems, with the following improvements with respect to the hitherto available models: (i) it does not rely on the hypothesis of local electro-neutrality, and allows to treat the salt-free case; (ii) the chemical exchange governing the partition between free and bound ions is properly taken into account; (iii) a fully analytical solution is provided. The variation of the frequency-dependent dielectric permittivity with both thermodynamic and kinetic characteristics of the free-bound ion equilibrium is presented. In particular, the relative weights of both relaxation modes (exchange and transport), and their characteristic frequencies are discussed. This study opens the way to the analysis of systems for which the usual models are irrelevant, such as salt-free clay gels or membranes.     

Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments

Carr-Purcell-Meiboom-Gill (CPMG) relaxation measurements employing trains of 180 degrees pulses with variable pulse spacing provide valuable information about systems undergoing millisecond-time-scale chemical exchange. Fits of the CPMG relaxation dispersion profiles yield rates of interconversion, relative populations, and absolute values of chemical shift differences between the exchanging states, |Deltaomega|. It is shown that the sign of Deltaomega that is lacking from CPMG dispersion experiments can be obtained from a comparison of chemical shifts in the indirect dimensions in either a pair of HSQC (heteronuclear single quantum coherence) spectra recorded at different magnetic fields or HSQC and HMQC (heteronuclear multiple quantum coherence) spectra obtained at a single field. The methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s(-1) at 25 degrees C.