CPMG Experiments Research Articles

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for (1)H spins in methyl (13)CHD2 groups, which improves the characterization of fast exchange processes. The influence of (1)H-(1)H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different (1)H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any (1)H relaxation dispersion of its methyl groups at 10 or 25°C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by (1)H and (13)C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

ShereKhan—calculating exchange parameters in relaxation dispersion data from CPMG experiments

Dynamics governing the function of biomolecule is usually described as exchange processes and can be monitored at atomic resolution with nuclear magnetic resonance (NMR) relaxation dispersion data. Here, we present a new tool for the analysis of CPMG relaxation dispersion profiles (ShereKhan). The web interface to ShereKhan provides a user-friendly environment for the analysis. A stable version of ShereKhan, the web application and documentation are available at http://sherekhan.bionmr.org. dole@nmr.mpibpc.mpg.de or mako@nmr.mpibpc.mpg.de.

Designing optimal universal pulses using second-order, large-scale, non-linear optimization

Recently, RF pulse design using first-order and quasi-second-order pulses has been actively investigated. We present a full second-order design method capable of incorporating relaxation, inhomogeneity in B0 and B1. Our model is formulated as a generic optimization problem making it easy to incorporate diverse pulse sequence features. To tame the computational cost, we present a method of calculating second derivatives in at most a constant multiple of the first derivative calculation time, this is further accelerated by using symbolic solutions of the Bloch equations. We illustrate the relative merits and performance of quasi-Newton and full second-order optimization with a series of examples, showing that even a pulse already optimized using other methods can be visibly improved.To be useful in CPMG experiments, a universal refocusing pulse should be independent of the delay time and insensitive of the relaxation time and RF inhomogeneity. We design such a pulse and show that, using it, we can obtain reliable R2 measurements for offsets within ±γB1. Finally, we compare our optimal refocusing pulse with other published refocusing pulses by doing CPMG experiments.

Studies on crude oil‐water biphasic mixtures by low‐field NMR

Low-field (1) H NMR was used in this work for the analysis of mixtures involving crude oils and water. CPMG experiments were performed to determine the transverse relaxation time (T2 ) distribution curves, which were computed by the inverse Laplace transform of the echo decay data. The instrument's ability of quantifying water and petroleum in biphasic mixtures following different methodologies was tested. For mixtures between deionized water and petroleum, one achieved excellent results, with root mean squared error of cross-validation (RMSECV) of 0.8% for a regression between the water content (wt %) and the relative area of the water peak in the T2 distribution curve, or a standard deviation of 0.9% for the relationship between the water content and the relative water peak area, corrected by the relative hydrogen index of the crude. In the case of biphasic mixtures of Mn(2+) -doped water and crude oils, the best result of RMSECV = 1.6% was achieved by using the raw magnetization decay data for a partial least squares regression.

Tracking Conformational Changes during Amyloidogenesis in Real-Time at Atomic-Resolution by NMR

Understanding the structural transitions that amyloid proteins undergo during amyloidogenesis would greatly enhance our understanding of this process. However, our knowledge has been currently largely limited to global conformational changes, with high-resolution structural information only available for the monomeric proteins and for a few structures of the final amyloid product. In particular, high-resolution structures of intermediate states have been notoriously absent with few exceptions. We show here that high-resolution structures of intermediates can be obtained by using SOFAST-HMQC, CPMG, NOESY, magic-angle-spinning, and other experiments in real-time to track the aggregation pathway at atomic-level detail and at a time-resolution of minutes. As examples, we show the aggregation pathways of Aβ and IAPP, two initially unstructured peptides implicated in Alzheimer's and type II Diabetes, respectively.While previous studies commonly show the Aβ1-40 is largely unstructured in solution before the formation of β-sheet oligomers, we show that Aβ1-40 gradually adopts a compact, partially folded helical structure over a period of several days. In this structure, the central hydrophobic region of the peptide forms a 310 helix from H13 to D23 and the N- and C-termini collapse against the helix due to the clustering of hydrophobic residues (pdb:2LFM). The formation of the helical intermediate is concentration dependent and can be partially reversed by dilution of the peptide. Helical intermediates have been predicted to be crucial on-pathway intermediates in amyloid fibrillogenesis, and the structure presented here presents a new target for structure-based intervention, shown here by the interaction of the helical intermediate with polyphenols. By contrast, the more amyloidogenic IAPP peptide shows only a gradual transition to the fiber form after an initial pH dependent formation of a micelle-like aggregate, with distinct β-sheet small oligomers forming only a small fraction of the observable population.

Multinuclear solid-state NMR of square-planar platinum complexes — Cisplatin and related systems

Multinuclear solid-state nuclear magnetic resonance (SSNMR) experiments have been performed on cisplatin and four related square-planar compounds. The wideband uniform rate smooth truncation – Carr–Purcell–Meiboom–Gill (WURST–CPMG) pulse sequence was utilized in NMR experiments to acquire 195Pt, 14N, and 35Cl ultra-wideline NMR spectra of high quality. Standard Hahn-echo and magic-angle spinning 195Pt NMR experiments are also performed to refine extracted chemical shielding (CS) tensor parameters. Platinum magnetic shielding (MS) tensor orientations are calculated using both plane-wave density functional theory (DFT) and standard DFT methods. The tensor orientations are shown to be highly constrained by molecular symmetry elements, but also influenced to some degree by intermolecular interactions. 14N WURST–CPMG experiments were performed on three compounds and electric field gradient (EFG) parameters (the quadrupolar coupling constant, CQ, and the asymmetry parameter, ηQ) are reported. First principles calculations of the 14N EFG tensor parameters and orientations and affirm their dependence on the local hydrogen bonding environment. 35Cl WURST–CPMG experiments on cisplatin and transplatin are reported, using two different static magnetic fields to extract EFG and CS tensor parameters, and 35Cl EFG tensor magnitudes and orientations are predicted using first principles calculations. Transverse (T2) relaxation data for all nuclei are used to investigate heteronuclear dipolar relaxation mechanisms, as well as the nature of the local hydrogen bonding environments.

Exact solution of the CPMG pulse sequence with phase variation down the echo train: Application to R2 measurements

An implicit exact algebraic solution of CPMG experiments is presented and applied to fit experiments. Approximate solutions are also employed to explore oscillations and effective decay rates of CPMG experiments. The simplest algebraic approximate solution has illustrated that measured intensities will oscillate in the conventional CPMG experiments and that using even echoes can suppress errors of measurements of R 2 due to the imperfection of high-power pulses. To deal with low-power pulses with finite width, we adapt the effective field to calculate oscillations. An optimization model with the effective field approximation and dimensionless variables is proposed to quantify oscillations of measured intensities of CPMG experiments of different phases of the π pulses. We show, as was known using other methods, that repeating one group of four pulses with different phases in CPMG experiments, which we call phase variation, but others call phase alternation or phase cycling, can significantly smooth the dependence of measured intensities on frequency offset in the range of ± 1 2 γ B 1 . In this paper, a second-order expression with respect to the ratio of frequency offset to π-pulse amplitude is developed to describe the effective R 2 of CPMG experiments when using a group phase variation scheme. Experiments demonstrate that (1) the exact calculation of CPMG experiments can remarkably eliminate systematic errors in measured R 2s due to the effects of frequency offset, even in the absence of phase variation; (2) CPMG experiments with group phase variation can substantially remove oscillations and effects of the field inhomogeneity; (3) the second-order expression of the effective decay rate with phase variation is able to provide reliable estimates of R 2 when offsets are roughly within± 1 2 γ B 1 ; and, most significantly, (4) the more sophisticated optimization model using an exact solution of the discretized CPMG experiment extends, to ± γB 1, the range of offsets for which reliable estimates of R 2 can be obtained when using the preferred phase variation scheme.

Hydrophobic Side Chain Dynamics of a Glutamate Receptor Ligand Binding Domain

Ionotropic glutamate receptors are ligand-gated ion channels that mediate much of the fast excitatory neurotransmission in the central nervous system. The extracellular ligand binding core (S1S2) of the GluR2 subtype of ionotropic glutamate receptors can be produced as a soluble protein with properties essentially identical to the corresponding domain in the intact, membrane-bound protein. Using a variety of biophysical techniques, much has been learned about the structure and dynamics of S1S2 and the relationship between its ligand-induced conformational changes and the function of the receptor. It is clear that dynamic processes are essential to the function of ionotropic glutamate receptors. We have isotopically labeled side chain methyls of GluR2 S1S2 and used NMR spectroscopy to study their dynamics on the ps-ns and mus-ms time scales. Increased disorder is seen in regions that are part of the key dimer interface in the intact protein. When glutamate is bound, the degree of ps-ns motion is less than that observed with other ligands, suggesting that the physiological agonist binds to a preformed binding site. At the slower time scales, the degree of S1S2 flexibility induced by ligand binding is greatest for willardiine partial agonists, least for antagonists, and intermediate for full agonists. Notable differences among bound ligands are in the region of the protein that forms a hinge between two lobes that close upon agonist binding, and along the beta-sheet in Lobe 2. These motions provide clues as to the functional properties of partial agonists and to the conformational changes associated with lobe closure and channel activation.

Binding Mechanism of an SH3 Domain Studied by NMR and ITC

Complexes between Src-homology 3 domains and proline-rich target peptides can have lifetimes on the order of milliseconds, making them too short-lived for kinetic characterization by conventional methods. Nuclear magnetic resonance (NMR) dynamics experiments are ideally suited to study such rapid binding equilibria, and additionally provide information on partly bound intermediate states. We used NMR together with isothermal titration calorimetry (ITC) to characterize the interaction of the SH3 domain from the Fyn tyrosine kinase with a 12-residue peptide at temperatures between 10 and 50 degrees C. NMR data at all temperatures are consistent with an effectively two-state binding reaction, such that any intermediates are either very weakly populated or exchange extremely rapidly with the free or bound forms. Dissociation rate constants, determined by CPMG and ZZ-exchange NMR experiments, range from k(off)(10 degrees C) = 4.5 s(-1) to k(off)(50 degrees C) = 331 s(-1). ITC data at all temperatures follow a simple two-state interaction model. Binding is favored enthalpically, with a dissociation enthalpy, DeltaH(D)(30 degrees C) = 15.4 kcal mol(-1), and disfavored entropically, with a dissociation entropy, DeltaS(D)(30 degrees C) = 20.0 cal mol(-1) K(-1). The free protein and peptide have significantly higher heat capacity than the bound complex, DeltaC(p) = 352 cal mol(-1) K(-1), which is consistent with the largely hydrophobic character of the binding interface. An Eyring plot of k(off) values gives an activation enthalpy of dissociation, DeltaH(D)(double dagger)(30 degrees C) = 19.3 kcal mol(-1) and exhibits slight curvature consistent with the ITC-derived value of DeltaC(p). The curvature suggests that nonpolar residues of the hydrophobic interface are solvated in the transition state for dissociation. Association rate constants were calculated as k(on) = k(off)/K(D), and range from k(on)(10 degrees C) = 1.03 x 10(8) M(-1) s(-1) to k(on)(50 degrees C) = 2.0 x 10(8) M(-1) s(-1), with an apparent activation enthalpy, DeltaH(A)(double dagger) = 3.4 kcal mol(-1). Both the magnitudes and temperature dependence of k(on) values are consistent with a diffusion-limited association mechanism. The combination of NMR and ITC data sheds light on how the Fyn tyrosine kinase is activated by binding to proline-rich targets, and represents a powerful approach for characterizing transient protein/ligand interactions.

Entropy-driven cAMP-dependent Allosteric Control of Inhibitory Interactions in Exchange Proteins Directly Activated by cAMP

Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices alpha1 and alpha2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.

Domain–domain motions in proteins from time-modulated pseudocontact shifts

In recent years paramagnetic NMR derived structural constraints have become increasingly popular for the study of biomolecules. Some of these are based on the distance and angular dependences of pseudo contact shifts (PCSs). When modulated by internal motions PCSs also become sensitive reporters on molecular dynamics. We present here an investigation of the domain-domain motion in a two domain protein (PA0128) through time-modulation of PCSs. PA0128 is a protein of unknown function from Pseudomonas aeruginosa (PA) and contains a Zn(2+) binding site in the N-terminal domain. When substituted with Co(2+) in the binding site, several resonances from the C-terminal domain showed severe line broadening along the (15)N dimension. Relaxation compensated CPMG experiments revealed that the dramatic increase in the (15)N linewidth came from contributions of chemical exchange. Since several sites with perturbed relaxation are localized to a single beta-strand region, and since extracted timescales of motion for the perturbed sites are identical, and since the magnitude of the chemical exchange contributions is consistent with PCSs, the observed rate enhancements are interpreted as the result of concerted domain motion on the timescale of a few milliseconds. Given the predictability of PCS differences and the easy interpretation of the experimental results, we suggest that these effects might be useful in the study of molecular processes occurring on the millisecond to microsecond timescale.

Strong coupling effects during X-pulse CPMG experiments recorded on heteronuclear ABX spin systems: artifacts and a simple solution

Simulation and experiment have been used to establish that significant artifacts can be generated in X-pulse CPMG relaxation dispersion experiments recorded on heteronuclear ABX spin-systems, such as (13)C( i )-(13)C( j )-(1)H, where (13)C( i ) and (13)C( j ) are strongly coupled. A qualitative explanation of the origin of these artifacts is presented along with a simple method to significantly reduce them. An application to the measurement of (1)H CPMG relaxation dispersion profiles in an HIV-2 TAR RNA molecule where all ribose sugars are protonated at the 2' position, deuterated at all other sugar positions and (13)C labeled at all sugar carbons is presented to illustrate the problems that strong (13)C-(13)C coupling introduces and a simple solution is proposed.

A Model for Agonism and Antagonism in an Ancient and Ubiquitous cAMP-binding Domain

The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R(p))-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S(p))-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R(p))- and (S(p))-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S(p))-cAMPS agonist from the (R(p))-cAMPS antagonist. Based on these data, we have proposed a model for the (R(p)/S(p))-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile(163) and Asp(170), respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.

Molecular mobility in model dough systems studied by time-domain nuclear magnetic resonance spectroscopy

The microscopic distribution and dynamic state of water and molecular mobility in various model systems are investigated using time-domain NMR spectroscopy. Starch and gluten showed different continuous distribution populations in T 21 (μsec range, obtained from One pulse experiments) and T 22 (msec range, obtained from CPMG experiments) proving that starch and gluten have different water dynamics and molecular mobility. A starch/gluten mixture (76:12, w/w) and wheat flour dough exhibited similar patterns indicating that water and molecular mobilities in dough tended to be more representative of interactions with starch than gluten, even though both water–starch and water–gluten interactions are occurring in wheat flour dough. Increasing the water content did not influence the continuous distribution pattern of T 21 but affected the relative amount of each fraction in T 21 (i.e. an increase of the more mobile fraction and a decrease of the less mobile fraction with increasing moisture). Added water has an important role in the more mobile fraction but not in the less mobile fraction, which is in μs range. This indicates that model food systems contain multiple microstructural domains with various water and molecular mobilities that show correspondingly different water dynamics. Therefore, the dispersion of various relaxation time constants helped identify the distribution of independent microstructural domains. The manipulation of the composition of the model food system influences the water dynamics and molecular mobility and provides a basis for the application of the microstructural domain concept to real food systems.

Mapping Polypeptide Self-Recognition through 1H Off-Resonance Relaxation

1H NMR relaxation rates provide a readily available and sensitive probe ideally suited to investigate the weak (KD approximately micromolar to millimolar range) interactions that frequently mediate polypeptide oligomerization in the early steps of amyloid fibrillogenesis. However, the measurement of transverse and longitudinal 1H relaxation rates is experimentally challenging due to J-transfer and selectivity problems in CPMG and inversion-recovery experiments, respectively. We show here that these problems are effectively circumvented by measuring nonselective off-resonance relaxation rates using an effective field tilted by 35.5 degrees . When applied to the Halpha spins of the Abeta (12-28) peptide, the proposed experiment provides a residue-resolution self-recognition map which is fully consistent with previous independent mutational studies. The method is anticipated to be widely applicable not only to the fast growing family of amyloidogenic peptides but also to the screening and mapping of protein-ligand interactions in general.

Conservation of μs−ms Enzyme Motions in the Apo- and Substrate-Mimicked State

Solution NMR spin-relaxation experiments were used to compare mus-ms dynamics in RNase A in the apo form and as complexed to the substrate-mimic, pTppAp. The crystal structure of the RNase A/pTppAp complex was determined and demonstrates that this ligand binds at the active site and utilizes established substrate binding sites in its interaction with RNase A. Relaxation-compensated CPMG experiments identify flexible residues in and around the active site in both the apo and pTppAp-bound enzyme. Quantitative analysis of the NMR spin-relaxation dispersion curves show that the time scale of motion in RNase A is unchanged when pTppAp binds and is similar to the time scale for the rate-determining step of the catalytic reaction. Temperature-dependent measurements provide an activation barrier for motion of 5.2 +/- 1.0 kcal/mol and 4.5 +/- 1.2 kcal/mol for the apo and pTppAp forms of RNase A, respectively. These data indicate very similar motion exists in the free and bound enzyme. Additionally, chemical shift data suggests that the magnitude of motion is also similar for these two forms and that it is likely that apo enzyme interconverts to a structure that resembles a ligand-bound form. Likewise, it appears that the bound conformation samples the apo enzyme form even when ligand is present. Taken together the data imply that RNase A is in a preexisting dynamic equilibrium between two conformations that represent the open and closed enzyme forms. These data suggest that ligand binding stabilizes the bound conformer but does not induce it.

Analysis of slow motions in the micro–millisecond range on domain 1 of annexin I

A preliminary study of the dynamics of the domain 1 of annexin I by Nuclear Magnetic Resonance is presented. The use of 15N-relaxation-compensated CPMG experiments allowed us to show the presence of conformational motions in the micro–millisecond range that extend over several segments along the domain. For 14 residues, exchange rate constants could be evaluated, and were found in the range 1300–2500 s –1. To cite this article: M. Gallopin et al., C.R. Chimie 00 (2004) .

Effects of chemical modification of wheat starch on molecular mobility as studied by pulsed 1H NMR

The effects of hydroxypropylation (0.05, 0.12, and 0.18 molar substitution (MS)) and cross-linking (0.03%, 0.1%, and 0.2%) on molecular mobility in low-moisture wheat starch were investigated by pulsed 1H NMR. Starch samples were equilibrated over an a w range of 0–0.93. Transverse relaxation ( T 2) times of the systems were obtained from single-pulse and CPMG experiments and were analysed by distributed exponential fitting. T 2s ∗ values associated with starch molecules increased with increasing MS of hydroxypropyl group, indicating increased mobility of starch chains. Proton relaxation was not significantly affected by cross-linking. The proton relaxation associated with water in control (alkali treated), hydroxypropylated and cross-linked starch showed two distinct populations for samples at a w between 0.43 and 0.93. In general, water mobility in both regions increased as a w or hydroxypropyl MS increased, while cross-linking at 0.03% and 0.1% produced little change in water mobility.

1H NMR studies of molecular mobility in wheat starch

Pulsed 1H NMR was used to determine moisture content and molecular mobility of wheat starch suspensions at water activity ( a w) values between 0 and 0.93. Moisture determination by NMR agreed well with gravimetric determinations ( R 2=0.99). Transverse proton relaxation ( T 2) of the systems were identified using single pulse and CPMG experiments. T 2 associated with starch molecules increased linearly from 7.1 to 10.1 μs as a w increased from 0 to 0.93, due to plasticizing effects of water. T 2 associated with water molecules showed a linear increase up to a w of 0.69. Results indicated that 1H NMR could be employed to determine moisture content of starch suspension and to identify and quantify the molecular motion of starch chains and water molecules of starch suspensions at low moisture levels.

NMR Investigation of the Dynamics of Tryptophan Side-chains in Hemoglobins

NMR relaxation measurements of 15N spin–lattice relaxation rate ( R 1), spin–spin relaxation rate ( R 2), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7 T and 14.1 T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or ( 2H, 15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (βD99N), rHb (αY42D/βD99N), and rHb (αV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (α14, β15, and β37) in Hb A for each αβ dimer. These Trp residues are located in important regions of the Hb molecule, i.e. α14Trp and β15Trp are located in the α 1β 1 subunit interface and β37Trp is located in the α 1β 2 subunit interface. The relaxation experiments show that amino acid substitutions in the α 1β 2 subunit interface can alter the dynamics of β37Trp. The transverse relaxation rate ( R 2) for β37Trp can serve as a marker for the dynamics of the α 1β 2 subunit interface. The relaxation parameters of deoxy-rHb Kemspey (βD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (αY42D/βD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY–CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of β37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure–function relationship in hemoglobin.