Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around approximately 33 ppm at pH 5.8 and 35 degrees C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to 1H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional 1H-15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D 1H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t(1) (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nzeta and 13Cepsilon (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.     

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.     

Comparison of methyl rotation axis order parameters derived from model-free analyses of (2)H and (13)C longitudinal and transverse relaxation rates measured in the same protein sample.

Recombinant HIV-1 protease was obtained from bacteria grown on a 98% D(2)O medium containing 3-(13)C pyruvic acid as the sole source of (13)C and (1)H. The purified protein is highly deuterated at non-methyl carbons, but contains significant populations of (13)CHD(2) and (13)CH(2)D methyl isotopomers. This pattern of isotope labeling permitted measurements of (1)H and (13)C relaxation rates of (13)CHD(2) isotopomers and (2)H (D) relaxation rates of (13)CH(2)D isotopomers using a single sample. The order parameters S(axis)(2), which characterize the motions of the methyl rotation axes, were derived from model-free analyses of R(1) and R(2) data sets measured for (13)C and (2)H spins. Our primary goal was to compare the S(axis)(2) values derived from the two independent types of data sets to test our understanding of the relaxation mechanisms involved. However, S(axis)(2) values derived from the analyses depend strongly on the geometry of the methyl group, the sizes of the quadrupolar and dipolar couplings, and the effects of bond vibrations and librations on these couplings. Therefore uncertainties in these basic physical parameters complicate comparison of the order parameters. This problem was circumvented by using an experimental relationship, between the methyl quadrupolar, (13)C-(13)C and (13)C-(1)H dipolar couplings, derived from independent measurements of residual static couplings of weakly aligned proteins by Ottiger and Bax (J. Am. Chem. Soc. 1999, 121, 4690-4695) and Mittermaier and Kay (J. Am. Chem. Soc. 1999, 121, 10608-10613). This approach placed a tight experimental restraint on the values of the (2)H quadrupolar and (13)C-(1)H dipolar interactions and greatly facilitated the accurate comparison of the relative values of the order parameters. When applied to our data this approach yielded satisfactory agreement between the S(axis)(2) values derived from the (13)C and (2)H data sets.     

Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy

A general method to enhance the sensitivity of the multidimensional NMR experiments performed at high-polarizing magnetic field via the significant reduction of the longitudinal proton relaxation times is described. The method is based on the use of two vast pools of "thermal bath" 1H spins residing on hydrogens covalently attached to carbon and oxygen atoms in 13C,15N labeled and fully protonated or fractionally deuterated proteins to uniformly enhance longitudinal relaxation of the 1HN spins and concomitantly the sensitivity of multipulse NMR experiments. The proposed longitudinal relaxation optimization is implemented in the 2D [15N,1H]-LTROSY, 2D [15N,1H]-LHSQC and 3D LTROSY-HNCA experiments yielding the factor 2-2.5 increase of the maximal signal-to-noise ratio per unit time at 600 MHz. At 900 MHz, the predicted decrease of the 1HN longitudinal relaxation times can be as large as one order of magnitude, making the proposed method an important tool for protein NMR at high magnetic fields.     

Nuclear magnetic relaxation in water revisited

In this study, we revisited nuclear magnetic relaxation of (1)H in water at very low Larmor frequencies that has been studied intensively in earlier years. We make use of the recently developed superconducting quantum interference device based ultra-low field NMR technique, which enables much easier access to the longitudinal spin-lattice relaxation time T(1) and the transversal spin-spin relaxation time T(2) below several kHz than traditional field cycling methods. Our data reproduce and complement the earlier results, in that they corroborate the finding of an exchange process with a correlation time of about 0.34 ms at room temperature which can be attributed to the migration of hydronium and hydroxyl ions in neutral water via hydrogen bridges. The corresponding relaxation process is driven by the interaction of the protons with (17)O and contributes to the T(1) and the T(2) relaxation rate by about 0.12 s(-1). In addition, we found evidence of a very slow exchange process at about 100 Hz that has hitherto not been reported.     

Mapping polypeptide self-recognition through (1)H 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.     

Broadband measurement of true transverse relaxation rates in systems with coupled protons: application to the study of conformational exchange

Accurate measurement of transverse relaxation rates in coupled spin systems is important in the study of molecular dynamics, but is severely complicated by the signal modulations caused by scalar couplings in spin echo experiments. The most widely used experiments for measuring transverse relaxation in coupled systems, CPMG and PROJECT, can suppress such modulations, but they also both suppress some relaxation contributions, and average relaxation rates between coupled spins. Here we introduce a new experiment which for the first time allows accurate broadband measurement of transverse relaxation rates of coupled protons, and hence the determination of exchange rate constants in slow exchange from relaxation measurements. The problems encountered with existing methods are illustrated, and the use of the new method is demonstrated for the classic case of hindered amide rotation and for the more challenging problem of exchange between helical enantiomers of a gold(i) complex.

Existing methods for measuring transverse relaxation give incorrect results in coupled spin systems. Measuring true relaxation rates extends their utility.     

Accuracy of SUPREX (stability of unpurified proteins from rates of H/D exchange) and MALDI mass spectrometry-derived protein unfolding free energies determined under non-EX2 exchange conditions

Described here is the impact of so-called non-EX2 exchange behavior on the accuracy of protein unfolding free energies (i.e., DeltaG u values) and m values (i.e.,-deltaDeltaG u/delta[denaturant] values) determined by an H/D exchange and mass spectrometry-based technique termed stability of unpurified proteins from rates of H/D exchange (SUPREX). Both experimental and theoretical results on a model protein, ubiquitin, reveal that reasonably accurate thermodynamic parameters for its folding reaction can be determined by SUPREX even when H/D exchange data is collected in a non-EX2 regime. Not surprisingly, the theoretical results reported here on a series of hypothetical protein systems with a wide range of biophysical properties show that the accuracy of SUPREX-derived DeltaG u and m values is compromised for many proteins when analyses are performed at high pH (e.g., pH 9) and for selected proteins with specific biophysical parameters (e.g., slow folding rates) when analyses are performed at lower pH. Of more significance is that the experimental and theoretical results reveal a means by which problems with non-EX2 exchange behavior can be detected in the SUPREX experiment without prior knowledge of the protein's biophysical properties. The results of this work also reveal that such problems with non-EX2 exchange behavior can generally be minimized if appropriate H/D exchange times are employed in the SUPREX experiment to yield SUPREX curve transition midpoints at chemical denaturant concentrations less than 2 M.     

Controlling the variation of axial water exchange rates in macrocyclic lanthanide(III) complexes

The rate of axial water exchange in well-defined series of lanthanide complexes depends on the extent of second sphere hydration which is determined by complex hydrophobicity and the nature of the lanthanide ion and its counter-ion.     

Water/polymer interactions in poly(amidoamine) hydrogels by 1H nuclear magnetic resonance relaxation and magnetization transfer

Hydrated cross-linked polymers belonging to the family of poly(amidoamine)s were investigated by high and low resolution (1)H nuclear magnetic resonance techniques in order to obtain information on water/polymer interactions in the swollen state. (1)H spin-spin and spin-lattice relaxation time analysis, as well as magnetization transfer experiments, indicated that water and polymer proton pools are essentially uncoupled, with water molecules diffusing fast within the hydrogel structure and exchanging between "bound" and free sites. For the polymer characterized by the highest cross-linking degree, there is strong evidence of a beadlike structure resulting in higher network rigidity and hydrogel micrometric heterogeneity.     

Deuterium spin probes of side-chain dynamics in proteins. 1. Measurement of five relaxation rates per deuteron in (13)C-labeled and fractionally (2)H-enriched proteins in solution

New pulse sequences are presented for the measurement of the relaxation of deuterium double quantum, quadrupolar order, and transverse antiphase magnetization in (13)CH(2)D methyl groups of (15)N-, (13)C-labeled, fractionally deuterated proteins. Together with previously developed experiments for measuring deuterium longitudinal and transverse decay rates [Muhandiram, D. R.; Yamazaki, T.; Sykes, B. D.; Kay, L. E. J. Am. Chem. Soc. 1995, 117, 11536], these schemes allow measurement of the five unique decay constants of a single deuteron, providing an unprecedented opportunity to investigate side-chain dynamics in proteins. All five deuterium relaxation rates have been measured for deuterons in the methyl groups of the B1 immunoglobulin binding domain of peptostreptococcal protein L and the N-terminal SH3 domain from the protein drk. Since values of the spectral density function at only three different frequencies contribute to the five relaxation rates, the self-consistency of the relaxation data is readily established. Very good agreement is obtained between calculated parameters describing the amplitudes and time scales of motion when different subsets of the relaxation data are employed.     

Chiral 1D Dy(III) compound showing slow magnetic relaxation

Herein, we present one chiral Dy(III) compound, namely Dy(L)(3)(1, HL = 2-methylbenzoic acid), synthesized from the achiral HL ligand. Within 1, along a direction the seven-coordinated Dy(III) ion are bridged by double L carboxylate and single L oxygen to give rise to the 1D helical chain. The magnetic studies suggest small intra-chain ferromagnetic interactions. Alternating current (AC) magnetic measurements show a frequency dependence of magnetic susceptibilities in both in-phase, χ', and out-of-phase, χ'. However, no obvious peak can be found even under 1000 Oe static field, suggesting the prevalence of quantum tunnelling effect at low temperature.     

C-13 magnetic relaxation rates and H-1 and C-13 paramagnetic shifts of Ni(II) complex of dopamine

The ¹H and ¹³C isotropic contact shifts and the ¹³C relaxation times of dopamine in aqueous solution have been measured in the presence of the Ni(II) ion. The pD dependence of the ¹H and ¹³C paramagnetic shifts was also investigated. From the analysis of the shifts at pD = 6.5 and from the INDO MO calculations on selected models of dopamine radicals, a dominant σ delocalization mechanism of the spin density is proposed. From the spin distribution on the ligand carbon atoms, the metal centered as well as the ligand centered dipolar contributions of the modified Solomon—Bloembergen equation were calculated and an estimate of the correlation time τ_c was given.     

Slow magnetic relaxation induced by a large transverse zero-field splitting in a Mn(II)Re(IV)(CN)2 single-chain magnet

The model compounds (NBu(4))(2)[ReCl(4)(CN)(2)] (1), (DMF)(4)ZnReCl(4)(CN)(2) (2), and [(PY5Me(2))(2)Mn(2)ReCl(4)(CN)(2)](PF(6))(2) (3) have been synthesized to probe the origin of the magnetic anisotropy barrier in the one-dimensional coordination solid (DMF)(4)MnReCl(4)(CN)(2) (4). High-field electron paramagnetic resonance spectroscopy reveals the presence of an easy-plane anisotropy (D > 0) with a significant transverse component, E, in compounds 1-3. These findings indicate that the onset of one-dimensional spin correlations within the chain compound 4 leads to a suppression of quantum tunneling of the magnetization within the easy plane, resulting in magnetic bistability and slow relaxation behavior. Within this picture, it is the transverse E term associated with the Re(IV) centers that determines the easy axis and the anisotropy energy scale associated with the relaxation barrier. The results demonstrate for the first time that slow magnetic relaxation can be achieved through optimization of the transverse anisotropy associated with magnetic ions that possess easy-plane anisotropy, thus providing a new direction in the design of single-molecule and single-chain magnets.     

Acetone and water on TiO2(110): H/D exchange

Isotopic H/D exchange between coadsorbed acetone and water on the TiO2(110) surface was examined using temperature programmed desorption (TPD) as a function of coverage and two surface pretreatments (O2 oxidation and mild vacuum reduction). Coadsorbed acetone and water interact repulsively on reduced TiO2(110) on the basis of results from the companion paper to this study, with water exerting a greater influence in destabilizing acetone and acetone having only a nominal influence on water. Despite the repulsive interaction between these coadsorbates, about 0.02 monolayers (ML) of a 1 ML d6-acetone on the reduced surface (vacuum annealed at 850 K to a surface oxygen vacancy population of 7%) exhibits H/D exchange with coadsorbed water, with the exchange occurring exclusively in the high-temperature region of the d6-acetone TPD spectrum at approximately 340 K. The effect was confirmed with combinations of d0-acetone and D2O. The extent of exchange decreased on the reduced surface for water coverages above approximately 0.3 ML due to the ability of water to displace coadsorbed acetone from first layer sites to the multilayer. In contrast, the extent of exchange increased by a factor of 3 when surface oxygen vacancies were pre-oxidized with O2 prior to coadsorption. In this case, there was no evidence for the negative influence of high water coverages on the extent of H/D exchange. Comparison of the TPD spectra from the exchange products (either d1- or d5-acetone depending on the coadsorption pairing) suggests that, in addition to the 340 K exchange process seen on the reduced surface, a second exchange process was observed on the oxidized surface at approximately 390 K. In both cases (oxidized and reduced), desorption of the H/D exchange products appeared to be reaction limited and to involve the influence of OH/OD groups (or water formed during recombinative desorption of OH/OD groups) instead of molecularly adsorbed water. The 340 K exchange process is assigned to reaction at step sites, and the 390 K exchange process is attributed to the influence of oxygen adatoms deposited during surface oxidation. The H/D exchange mechanism likely involves an enolate or propenol surface intermediate formed transiently during the desorption of oxygen-stabilized acetone molecules.     

Room-temperature icelike water in kanemite detected by 2H NMR T1 relaxation

2H NMR was used to study the nature of deuterated water in kanemite. Evidence is presented that shows that the water changes state from liquid to solid at room temperature during the hydration reaction that forms kanemite. The deuterium nuclei in the water experience rapid tetrahedral jumps in a hydrogen-bonded lattice like those observed in 2H2O ice.