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.     

An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange

A series of experiments are presented that provide an exchange-free measure of dipole-dipole (15)N transverse relaxation, R(dd), that can then be substituted for (15)N R(1rho) or R(2) rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving (1)H-(15)N longitudinal order, anti-phase (1)H and (15)N single-quantum coherences, and (1)H-(15)N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that R(dd) values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1-2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b(562) that shows large contributions to (15)N R(1rho) relaxation from chemical exchange.     

Probing aerogels by multiple quantum filtered (131)Xe NMR spectroscopy.

At the interface between solid surfaces and cavities filled with gaseous or liquid xenon, the nuclear magnetization of (131)Xe (S = (3)/(2)) is subject to quadrupolar interactions which may lead to higher rank single-quantum coherences that can be described by tensor elements T(2,)(+/-)(1) and T(3,)(+/-)(1). This can be demonstrated by multiple-quantum filtered (MQF) NMR experiments. In gaseous xenon on Pyrex surfaces, the primary source of such coherences was shown to be coherent evolution induced by a nonvanishing average quadrupolar coupling. In this contribution, MQF NMR is applied to aerogels filled with liquid xenon to demonstrate the potential of this technique for material sciences. Xenon in the liquid phase provides a sufficient spin density to obtain reasonable signal-to-noise ratios. Coherent evolution and relaxation both contribute to the creation of higher rank coherences depending on the presence or absence of water molecules on the surface. These two processes can be distinguished experimentally and provide complementary information about the surface of the host material.     

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.     

Polychromatic selective population inversion for TROSY experiments with large proteins

This paper presents polychromatic selective polarization inversion (PC-SPI) as an alternative to the polarization transfer methods recently developed for the application of NMR to large biological molecules. Theoretical and numerical considerations indicate that PC-SPI has the potential for more efficient polarization transfer under conditions of rapid transverse relaxation compared to J coupling- and cross-correlated relaxation-based transfers. The main advantage offered by the method presented here is the maintenance of near-optimal trajectories of inversion of the individual components of the spin magnetization while using broadband optimized pulses. A 2D experiment was implemented combining PC-SPI with TROSY-based chemical shift correlation. The experiment was applied to detect (15)N-(1)H chemical shift correlation spectra of a 200 kDa complex consisting of an 80% (2)H- and uniformly (15)N,(13)C-labeled 22 kDa portion of complement receptor type 1 and unlabeled C3b of complement (180 kDa).     

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.     

TROSY-based z-exchange spectroscopy: application to the determination of the activation energy for intermolecular protein translocation between specific sites on different DNA molecules

A two-dimensional TROSY-based z-exchange 1H-15N correlation experiment for the quantitative analysis of kinetic processes in the slow exchange regime is presented. The pulse scheme converts the product operator terms Nz into 2NzHz and 2NzHz into -Nz in the middle of the z-mixing period, thereby suppressing the buildup of spurious semi-TROSY peaks arising from the different relaxation rates for the Nz and 2NzHz terms and simplifying the behavior of longitudinal magnetization for an exchanging system during the mixing period. Theoretical considerations and experimental data demonstrate that the TROSY-based z-exchange experiment permits quantitative determination of rate constants using the same procedure as that for the conventional non-TROSY 15Nz-exchange experiment. Line narrowing as a consequence of the use of the TROSY principle makes the method particularly suitable for kinetic studies at low temperature, thereby permitting activation energies to be extracted from data acquired over a wider temperature range. We applied this method to the investigation of the process whereby the HoxD9 homeodomain translocates between specific target sites on different DNA molecules via a direct transfer mechanism without going through the intermediary of free protein. The activation enthalpy for intermolecular translocation was determined to be 17 kcal/mol.     

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.     

An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins

A new (15)N constant-time relaxation dispersion pulse scheme for the quantification of millisecond time-scale exchange dynamics in proteins is presented. The experiment differs from previously developed sequences in that it includes (1)H continuous-wave decoupling during the (15)N Carr-Purcell-Meiboom-Gill (CPMG) pulse train that significantly improves the relaxation properties of (15)N magnetization, leading to sensitivity gains in experiments. Moreover, it is shown that inclusion of an additional (15)N 180 degrees refocusing pulse (phase cycled +/- x) in the center of the CPMG pulse train, consisting of 1(5)N 180 degrees (y) pulses, provides compensation for pulse imperfections beyond the normal CPMG scheme. Relative to existing relaxation-compensated constant-time relaxation dispersion pulse schemes, nu(CPMG) values that are only half as large can be employed, offering increased sensitivity to slow time-scale exchange processes. The robustness of the methodology is illustrated with applications involving a pair of proteins: an SH3 domain that does not show millisecond time-scale exchange and an FF domain with significant chemical exchange contributions.     

Detection of multiple-quantum coherences with projective nuclear magnetic resonance measurement

It is shown that in nuclear magnetic resonance, multiple-quantum (MQ) coherences can be detected "instantly" by exploiting the principle of quantum-mechanical projective measurement. Therefore, the mixing period, which involves collective multispin dynamics and converts MQ coherences into observable single-quantum coherence (magnetization), is not necessary. The experimental examples are given for two finite clusters: benzene in liquid crystal and liquid crystal 4'-n-pentyl-4-cyanobiphenyl, and for solid adamantane with an infinite network of dipolar couplings.     

Solution NMR techniques for large molecular and supramolecular structures

Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa. With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N-signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H. This paper describes the experimental schemes used and details of their setup for individual measurements. The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines. Optimal transfer times for 2D [(15)N,(1)H]-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa. These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure. The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s. Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually.     

Off-resonance TROSY (R1 rho - R1) for quantitation of fast exchange processes in large proteins

Current solution NMR experiments for characterizing conformational exchange processes in large proteins are limited to exchange rates ca. 500-3000 s-1. A TROSY-based constant relaxation time (R1rho - R1) experiment is designed to extend this capability to measure motion with rates up to 105 s-1 in large macromolecules. The experiment combines off-resonance spin-lock rf fields, which provide access to the faster time-scale dynamics, with TROSY coherence selection, which extends the molecular-weight range available for study. When implemented on the 53-kDa dimeric enzyme triosephosphate isomerase, the experiment yielded substantial gains in signal-to-noise (up to 60%) over current experiments at modest static magnetic fields (14.1 T). The TROSY (R1rho - R1) experiment should therefore be of general utility for investigation of fast conformational exchange events in large proteins.     

Slow magnetic relaxation of a ferromagnetic Ni(II)5 cluster with an S = 5 ground state

Complex [Ni 5{pyCOpyC(O)(OMe)py} 2(O 2CMe) 4(N 3) 4(MeOH) 2].2MeOH.2.6H 2O ( 1.2MeOH.2.6H 2O) was synthesized by the reaction of Ni(O 2CMe) 2.4H 2O with pyCOpyCOpy and NaN 3 in refluxing MeOH. It crystallizes in the monoclinic C2/ c space group and consists of five Ni (II) atoms in a helical arrangement. Direct current magnetic susceptibility studies reveal ferromagnetic interactions between the Ni (II) ( S = 1) ions, stabilizing an S = 5 ground state. Alternating current susceptibility experiments revealed the existence of out-of-phase signals indicative of slow magnetic relaxation. Analysis of the signals showed that they are composite, suggesting more than one relaxation process, while analysis of their magnitudes suggests not all molecules undergo slow magnetic relaxation. Magnetization field-sweep experiments revealed hysteresis at 1.8 K, and magnetization decay experiments clearly verified the appearance of slow magnetic relaxation at that temperature.     

Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies

Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73((13)C,(15)N)/74-108((15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N-(13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H-(15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108((15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these 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.     

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.     

Eta(z)/kappa: a transverse relaxation optimized spectroscopy NMR experiment measuring longitudinal relaxation interference

NMR spin relaxation experiments provide a powerful tool for the measurement of global and local biomolecular rotational dynamics at subnanosecond time scales. Technical limitations restrict most spin relaxation studies to biomolecules weighing less than 10 kDa, considerably smaller than the average protein molecular weight of 30 kDa. In particular, experiments measuring eta(z), the longitudinal (1)H(N)-(15)N dipole-dipole (DD)/(15)N chemical shift anisotropy (CSA) cross-correlated relaxation rate, are among those least suitable for use with larger biosystems. This is unfortunate because these experiments yield valuable insight into the variability of the (15)N CSA tensor over the polypeptide backbone, and this knowledge is critical to the correct interpretation of most (15)N-NMR backbone relaxation experiments, including R(2) and R(1). In order to remedy this situation, we present a new (1)H(N)-(15)N transverse relaxation optimized spectroscopy experiment measuring eta(z) suitable for applications with larger proteins (up to at least 30 kDa). The presented experiment also yields kappa, the site-specific rate of longitudinal (1)H(N)-(1)H(') DD cross relaxation. We describe the eta(z)/kappa experiment's performance in protonated human ubiquitin at 30.0 degrees C and in protonated calcium-saturated calmodulin/peptide complex at 20.0 degrees C, and demonstrate preliminary experimental results for a deuterated E. coli DnaK ATPase domain construct at 34 degrees C.     

Determination of Na+ binding parameters by relaxation analysis of selected 23Na NMR coherences: RNA, BSA and SDS

Nuclear magnetic resonance provides several unique means of investigating the interactions between different inorganic ions and various macromolecules. (23)Na is a quadrupolar nucleus, meaning that relaxation analysis of the various coherences allows the measurement of its binding to biological macromolecules. In this study, we analyzed the quadrupolar relaxation of (23)Na(+) longitudinal magnetization and single- and triple-quantum coherences in aqueous systems containing RNA, bovine serum albumin and sodium dodecyl sulfate micelles. The effectiveness of the James-Noggle method for determining binding constants was evaluated in these systems, and also the applicability of various (23)Na coherences in providing information on the extent and affinity of binding to the three different classes of biomolecules.     

A new spin probe of protein dynamics: nitrogen relaxation in 15N-2H amide groups

(15)N spin relaxation data have provided a wealth of information on protein dynamics in solution. Standard R(1), R(1)(rho), and NOE experiments aimed at (15)N[(1)H] amide moieties are complemented in this work by HA(CACO)N-type experiments allowing the measurement of nitrogen R(1) and R(1)(rho) rates at deuterated (15)N[(2)D] sites. Difference rates obtained using this approach, R(1)((15)N[(1)H]) - R(1)((15)N[(2)D]) and R(2)((15)N[(1)H]) - R(2)((15)N[(2)D]), depend exclusively on dipolar interactions and are insensitive to (15)N CSA and R(ex) relaxation mechanisms. The methodology has been tested on a sample of peptostreptococcal protein L (63 residues) prepared in 50% H(2)O-50% D(2)O solvent. The results from the new and conventional experiments are found to be consistent, with respect to both local backbone dynamics and overall protein tumbling. Combining several data sets permits evaluation of the spectral density J(omega(D) + omega(N)) for each amide site. This spectral density samples a uniquely low frequency (26 MHz at a 500 MHz field) and, therefore, is expected to be highly useful for characterizing nanosecond time scale local motions. The spectral density mapping demonstrates that, in the case of protein L, J(omega(D) + omega(N)) values are compatible with the Lipari-Szabo interpretation of backbone dynamics based on the conventional (15)N relaxation data.