Probing slow time scale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: application to methionine residues in a cavity mutant of T4 lysozyme.

A relaxation dispersion-based NMR experiment is presented for the measurement and quantitation of micros-ms dynamic processes at methyl side-chain positions in proteins. The experiment measures the exchange contribution to the 13C line widths of methyl groups using a constant-time CPMG scheme. The effects of cross-correlated spin relaxation between dipole-dipole and dipole-CSA interactions as well as the effects of scalar coupling responsible for mixing of magnetization modes during the course of the experiment have been investigated in detail both theoretically and through simulations. It is shown that the complex relaxation properties of the methyl spin system do not complicate extraction of accurate exchange parameters as long as care is taken to ensure that appropriate magnetization modes are interchanged in the middle of the constant-time CPMG period. An application involving the measurement of relaxation dispersion profiles of methionine residues in a Leu99Ala substitution of T4 lysozyme is presented. All of the methionine residues are sensitive to an exchange event with a rate on the order of 1200 s(-1) at 20 degrees C that may be linked to a process in which hydrophobic ligands are able to rapidly bind to the cavity that is present in this mutant.     

Double- and zero-quantum NMR relaxation dispersion experiments sampling millisecond time scale dynamics in proteins

TROSY-based NMR relaxation dispersion experiments that measure the decay of double- and zero-quantum (1)H-(15)N coherences as a function of applied (1)H and (15)N radio frequency (rf) fields are presented for studying millisecond dynamic processes in proteins. These experiments are complementary to existing approaches that measure dispersions of single-quantum (15)N and (1)H magnetization. When combined, data from all four coherences provide a more quantitative picture of dynamics, making it possible to distinguish, for example, between two-site and more complex exchange processes. In addition, a TROSY-based pulse scheme is described for measuring the relaxation of amide (1)H single-quantum magnetization, obtained by a simple modification of the multiple-quantum experiments. The new methodology is applied to a point mutant of the Fyn SH3 domain that exchanges between folded and unfolded states at 25 degrees C.     

Multiple-quantum relaxation dispersion NMR spectroscopy probing millisecond time-scale dynamics in proteins: theory and application

New relaxation dispersion experiments are presented that probe millisecond time-scale dynamical processes in proteins. The experiments measure the relaxation of (1)H-(15)N multiple-quantum coherence as a function of the rate of application of either (1)H or (15)N refocusing pulses during a constant time relaxation interval. In contrast to the dispersion profiles generated from more conventional (15)N((1)H) single-quantum relaxation experiments that depend on changes in (15)N((1)H) chemical shifts between exchanging states, (1)H-(15)N multiple-quantum dispersions are sensitive to changes in the chemical environments of both (1)H and (15)N spins. The resulting multiple-quantum relaxation dispersion profiles can, therefore, be quite different from those generated by single-quantum experiments, so that an analysis of both single- and multiple-quantum profiles together provides a powerful approach for obtaining robust measures of exchange parameters. This is particularly the case in applications to protonated proteins where other methods for studying exchange involving amide proton spins are negatively influenced by contributions from neighboring protons. The methodology is demonstrated on protonated and perdeuterated samples of a G48M mutant of the Fyn SH3 domain that exchanges between folded and unfolded states in solution.     

Divided-evolution-based pulse scheme for quantifying exchange processes in proteins: powerful complement to relaxation dispersion experiments

A method for quantifying millisecond time scale exchange in proteins is presented based on scaling the rate of chemical exchange using a 2D (15)N, (1)H(N) experiment in which (15)N dwell times are separated by short spin-echo pulse trains. Unlike the popular Carr-Purcell-Meiboom-Gill (CPMG) experiment where the effects of a radio frequency field on measured transverse relaxation rates are quantified, the new approach measures peak positions in spectra that shift as the effective exchange time regime is varied. The utility of the method is established through an analysis of data recorded on an exchanging protein-ligand system for which the exchange parameters have been accurately determined using alternative approaches. Computations establish that a combined analysis of CPMG and peak shift profiles extends the time scale that can be studied to include exchanging systems with highly skewed populations and exchange rates as slow as 20 s(-1).     

Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.     

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.     

Off-resonance R(1rho) NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain

An (15)N NMR R(1rho) relaxation experiment is presented for the measurement of millisecond time scale exchange processes in proteins. On- and off-resonance R(1rho) relaxation profiles are recorded one residue at a time using a series of one-dimensional experiments in concert with selective Hartmann-Hahn polarization transfers. The experiment can be performed using low spin-lock field strengths (values as low as 25 Hz have been tested), with excellent alignment of magnetization along the effective field achieved. Additionally, suppression of the effects of cross-correlated relaxation between dipolar and chemical shift anisotropy interactions and (1)H-(15)N scalar coupled evolution is straightforward to implement, independent of the strength of the (15)N spin-locking field. The methodology is applied to study the folding of a G48M mutant of the Fyn SH3 domain that has been characterized previously by CPMG dispersion experiments. It is demonstrated through experiment that off-resonance R(1rho) data measured at a single magnetic field and one or more spin-lock field strengths, with amplitudes on the order of the rate of exchange, allow a complete characterization of a two-site exchange process. This is possible even in the case of slow exchange on the NMR time scale, where complementary approaches involving CPMG-based experiments fail. Advantages of this methodology in relation to other approaches are described.     

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.     

Separating degenerate (1)H transitions in methyl group probes for single-quantum (1)H-CPMG relaxation dispersion NMR spectroscopy

A relaxation dispersion pulse scheme is presented for quantifying chemical exchange processes in proteins that exploits 1H chemical shifts as probes of changes in conformation. The experiment selects 1H single-quantum magnetization from the I = 1/2 manifolds of the methyl group, which behave like AX spin systems, while suppressing coherences that derive from the 3/2 manifold that are extremely sensitive to pulse imperfections and that would otherwise severely compromise the accuracy of the experiment. The utility of the sequence is first demonstrated with an application to a protein system that is known not to undergo chemical exchange and flat dispersion profiles are obtained. Subsequently, the methodology is applied to study the folding of a G48M mutant of the Fyn SH3 domain that has been shown previously to undergo exchange between folded and unfolded states on the millisecond time scale.     

Quantifying millisecond exchange dynamics in proteins by CPMG relaxation dispersion NMR using side-chain 1H probes

A Carr-Purcell-Meiboom-Gill relaxation dispersion experiment is presented for quantifying millisecond time-scale chemical exchange at side-chain (1)H positions in proteins. Such experiments are not possible in a fully protonated molecule because of magnetization evolution from homonuclear scalar couplings that interferes with the extraction of accurate transverse relaxation rates. It is shown, however, that by using a labeling strategy whereby proteins are produced using {(13)C,(1)H}-glucose and D(2)O a significant number of 'isolated' side-chain (1)H spins are generated, eliminating such effects. It thus becomes possible to record (1)H dispersion profiles at the β positions of Asx, Cys, Ser, His, Phe, Tyr, and Trp as well as the γ positions of Glx, in addition to the methyl side-chain moieties. This brings the total of amino acid side-chain positions that can be simultaneously probed using a single (1)H dispersion experiment to 16. The utility of the approach is demonstrated with an application to the four-helix bundle colicin E7 immunity protein, Im7, which folds via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. The extracted (1)H chemical shift differences at side-chain positions provide valuable restraints in structural studies of invisible, excited states, complementing backbone chemical shifts that are available from existing relaxation dispersion experiments.     

Multiple-site exchange in proteins studied with a suite of six NMR relaxation dispersion experiments: an application to the folding of a Fyn SH3 domain mutant

The three-site exchange folding reaction of an (15)N-labeled, highly deuterated Gly48Met mutant of the Fyn SH3 domain has been characterized at 25 degrees C using a suite of six CPMG-type relaxation dispersion experiments that measure exchange contributions to backbone (1)H and (15)N transverse relaxation rates in proteins. It is shown that this suite of experiments allows the extraction of all the parameters of this multisite exchange process in a robust manner, including chemical shift differences between exchanging states, from a data set recorded at only a single temperature. The populations of the exchanging folded, intermediate, and unfolded states that are fit are 94, 0.7, and 5%, respectively. Despite the small fraction of the intermediate, structural information is obtained for this state that is consistent with the picture of SH3 domain folding that has emerged from other studies. Taken together, the six dispersion experiments facilitate the complete reconstruction of (1)H-(15)N correlation spectra for the unfolded and intermediate states that are "invisible" in even the most sensitive of NMR experiments.     

Characterization of specific protein association by 15N CPMG relaxation dispersion NMR: the GB1(A34F) monomer-dimer equilibrium

The Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion NMR experiment is a powerful means for detecting and characterizing conformational exchange. This experiment reports the exchange of chemical shifts and therefore can monitor all chemical exchange phenomena, not only intramolecular conformational exchange. Here, we report a CPMG transverse relaxation dispersion study for the monomer-dimer equilibrium of the GB1 point mutant, Ala-34-Phe (GB1(A34F)). This variant exists predominantly as a side-by-side dimer at high concentration (>1 mM). We demonstrate that the dispersion experiment is exceptionally valuable for studying association equilibria since it is extremely sensitive to the minor population in the equilibrium. Twenty-eight individual amide sites in the GB1(A34F) dimer protein were monitored via a 2D (15)N-(1)H HSQC spectroscopy, and all relaxation-derived data are consistent with predominantly an exchange process between dimer and monomer species.     

Communication: Phase incremented echo train acquisition in NMR spectroscopy

We present an improved and general approach for implementing echo train acquisition (ETA) in magnetic resonance spectroscopy, particularly where the conventional approach of Carr-Purcell-Meiboom-Gill (CPMG) acquisition would produce numerous artifacts. Generally, adding ETA to any N-dimensional experiment creates an N + 1 dimensional experiment, with an additional dimension associated with the echo count, n, or an evolution time that is an integer multiple of the spacing between echo maxima. Here we present a modified approach, called phase incremented echo train acquisition (PIETA), where the phase of the mixing pulse and every other refocusing pulse, φ(P), is incremented as a single variable, creating an additional phase dimension in what becomes an N + 2 dimensional experiment. A Fourier transform with respect to the PIETA phase, φ(P), converts the φ(P) dimension into a Δp dimension where desired signals can be easily separated from undesired coherence transfer pathway signals, thereby avoiding cumbersome or intractable phase cycling schemes where the receiver phase must follow a master equation. This simple modification eliminates numerous artifacts present in NMR experiments employing CPMG acquisition and allows "single-scan" measurements of transverse relaxation and J-couplings. Additionally, unlike CPMG, we show how PIETA can be appended to experiments with phase modulated signals after the mixing pulse.     

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.     

Disulfide bond isomerization in basic pancreatic trypsin inhibitor: multisite chemical exchange quantified by CPMG relaxation dispersion and chemical shift modeling

Conformational changes occurring on the microsecond-millisecond time scale in basic pancreatic trypsin inhibitor (BPTI) are investigated using nuclear magnetic resonance spectroscopy. The rczz CPMG experiment (Wang, C.; Grey, M. J.; Palmer, A. G. J. Biomol. NMR 2001, 21, 361-366) is used to record (15)N spin relaxation dispersion data, R(ex)(1/tau(cp)), in which 1/tau(cp) is the pulsing rate in the CPMG sequence, at two static magnetic fields, 11.7 and 14.1 T, and three temperatures, 280, 290, and 300 K. These data are used to characterize the kinetics and mechanism of chemical exchange line broadening of the backbone (15)N spins of Cys 14, Lys 15, Cys 38, and Arg 39 in BPTI. Line broadening is found to result from two processes: the previously identified isomerization of the Cys 38 side chain between chi(1) rotamers (Otting, G.; Liepinsh, E.; Wüthrich, K. Biochemistry 1993, 32, 3571-3582) and a previously uncharacterized process on a faster time scale. At 300 K, both processes contribute significantly to the relaxation dispersion for Cys 14 and an analytical expression for a linear three-site exchange model is used to analyze the data. At 280 K, isomerization of the Cys 38 side chain is negligibly slow and the faster process dominates the relaxation dispersion for all four spins. Global analysis of the temperature and static field dependence of R(ex)(1/tau(cp)) for Cys 14 and Lys 15 is used to determine the activation parameters and chemical shift changes for the previously uncharacterized chemical exchange process. Through an analysis of a database of chemical shifts, (15)N chemical shift changes for Cys 14 and Lys 15 are interpreted to result from a chi(1) rotamer transition of Cys 14 that converts the Cys 14-Cys 38 disulfide bond between right- and left-handed conformations. At 290 K, isomerization of Cys 14 occurs with a forward and reverse rate constant of 35 s(-1) and 2500 s(-1), respectively, a time scale more than 30-fold faster than the Cys 38 chi(1) isomerization. A comparison of the kinetics and thermodynamics for the transitions between the two alternative Cys 14-Cys 38 conformations highlights the factors that affect the contribution of disulfide bonds to protein stability.     

Rapid Determination of Fast Protein Dynamics from NMR Chemical Exchange Saturation Transfer Data

Functional motions of ¹⁵N‐labeled proteins can be monitored by solution NMR spin relaxation experiments over a broad range of timescales. These experiments however typically take of the order of several days to a week per protein. Recently, NMR chemical exchange saturation transfer (CEST) experiments have emerged to probe slow millisecond motions complementing R_1ρ and CPMG‐type experiments. CEST also simultaneously reports on site‐specific R₁ and R₂ parameters. It is shown here how CEST‐derived R₁ and R₂ relaxation parameters can be measured within a few hours at an accuracy comparable to traditional relaxation experiments. Using a “lean” version of the model‐free approach S² order parameters can be determined that match those from the standard model‐free approach applied to ¹⁵N R₁, R₂, and {¹H}‐¹⁵N NOE data. The new methodology, which is demonstrated for ubiquitin and arginine kinase (42 kDa), should serve as an effective screening tool of protein dynamics from picosecond‐to‐millisecond timescales.     

Conformational Dynamics in the Core of Human Y145Stop Prion Protein Amyloid Probed by Relaxation Dispersion NMR

Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using ¹⁵N rotating frame (R_1ρ) relaxation dispersion solid-state nuclear magnetic resonance spectroscopy over a wide range of spin-lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two-state exchange process with a common exchange rate of 1000 s⁻¹, corresponding to protein backbone motion on the timescale of 1 ms, and an excited-state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited-state populations (∼5–15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100–300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.     

Mapping chemical exchange in proteins with MW > 50 kD

Chemical exchange reveals motions in proteins that are critical for ligand binding, catalysis, and allosteric regulation at the microsecond to millisecond time scale. The detection of chemical exchange is inherently difficult in large proteins because of the fast transverse relaxation rate (R2) and spectral overlap. Here we report novel pulse sequences for the rapid identification of chemical exchange applicable to large deuterated proteins with MW greater than 30 kD. The success of our method is demonstrated in triosephosphate isomerase (TIM, MW = 54 kD).     

Microsecond time-scale conformational exchange in proteins: using long molecular dynamics trajectory to simulate NMR relaxation dispersion data

With the advent of ultra-long MD simulations it becomes possible to model microsecond time-scale protein dynamics and, in particular, the exchange broadening effects (R(ex)) as probed by NMR relaxation dispersion measurements. This new approach allows one to identify the exchanging species, including the elusive "excited states". It further helps to map out the exchange network, which is potentially far more complex than the commonly assumed 2- or 3-site schemes. Under fast exchange conditions, this method can be useful for separating the populations of exchanging species from their respective chemical shift differences, thus paving the way for structural analyses. In this study, recent millisecond-long MD trajectory of protein BPTI (Shaw et al. Science 2010, 330, 341) is employed to simulate the time variation of amide (15)N chemical shifts. The results are used to predict the exchange broadening of (15)N lines and, more generally, the outcome of the relaxation dispersion measurements using Carr-Purcell-Meiboom-Gill sequence. The simulated R(ex) effect stems from the fast (~10-100 μs) isomerization of the C14-C38 disulfide bond, in agreement with the prior experimental findings (Grey et al. J. Am. Chem. Soc. 2003, 125, 14324).