[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.     

The effect of noncollinearity of 15N-1H dipolar and 15N CSA tensors and rotational anisotropy on 15N relaxation, CSA/dipolar cross correlation, and TROSY

Current approaches to 15N relaxation in proteins assume that the 15N-1H dipolar and 15N CSA tensors are collinear. We show theoretically that, when there is significant anisotropy of molecular rotation, different orientations of the two tensors, experimentally observed in proteins, nucleic acids, and small peptides, will result in differences in site- specific correlation functions and spectral densities. The standard treatments of the rates of longitudinal and transverse relaxation of amide 15N nuclei, of the 15N CSA/15N-1H dipolar cross correlation, and of the TROSY experiment are extended to account for the effect of noncollinearity of the 15N-1H dipolar and 15N CSA (chemical shift anisotropy) tensors. This effect, proportional to the degree of anisotropy of the overall motion, (D/D–1), is sensitive to the relative orientation of the two tensors and to the orientation of the peptide plane with respect to the diffusion coordinate frame. The effect is negligible at small degrees of anisotropy, but is predicted to become significant for D/D1.5, and at high magnetic fields. The effect of noncollinearity of 15N CSA and 15N-1H dipolar interaction is sensitive to both gross (hydrodynamic) properties and atomic-level details of protein structure. Incorporation of this effect into relaxation data analysis is likely to improve both precision and accuracy of the derived characteristics of protein dynamics, especially at high magnetic fields and for molecules with a high degree of anisotropy of the overall motion. The effect will also make TROSY efficiency dependent on local orientation in moderately anisotropic systems.     

A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases

The microtubule-associated protein tau is impacted in neurodegeneration and dementia through its deposition in the form of paired helical filaments in Alzheimer's disease neurofibrillary tangles and through mutations linking it to the autosomal dominant disorder frontotemporal dementia with Parkinsonism. When isolated in solution tau is intrinsically unstructured and does not fold, while the conformation of the protein in the microtubule-bound state remains uncharacterized. Here we show that the repeat region of tau, which has been reported both to mediate tau microtubule interactions and to constitute the proteolysis-resistant core of disease-associated tau aggregates, associates with lipid micelles and vesicles and folds into an ordered structure upon doing so. In addition to providing the first structural insights into a folded state of tau, our results support a role for lipid membranes in mediating tau function and tau pathology.     

The use of TROSY for detection and suppression of conformational exchange NMR line broadening in biological macromolecules

The interference between conformational exchange-induced time-dependent variations of chemical shifts in a pair of scalar coupled ¹H and ¹⁵N spins is used to construct novel TROSY-type NMR experiments to suppress NMR signal loss in [¹⁵N,¹H]-correlation spectra of a 14-mer DNA duplex free in solution and complexed with the Antp homeodomain. An analysis of double- and zero-quantum relaxation rates of base ¹H–¹⁵N moieties showed that for certain residues the contribution of conformational exchange-induced transverse relaxation might represent a dominant relaxation mechanism, which, in turn, can be effectively suppressed by TROSY. The use of the new TROSY method for exchange-induced transverse relaxation optimization is illustrated with two new experiments, 2D ^h1 J _HN,^h2 J _NN-quantitative [¹⁵N,¹H]-TROSY to measure ^h1 J _HN and ^h2 J _NN scalar coupling constants across hydrogen bonds in nucleic acids, and 2D (^h2 J _NN+^h1 J _NH)-correlation-[¹⁵N,¹H]-TROSY to correlate ¹H^N chemical shifts of bases with the chemical shifts of the tertiary ¹⁵N spins across hydrogen bonds using the sum of the trans-hydrogen bond coupling constants in nucleic acids.     

Gradient and sensitivity enhancement of 2D TROSY with water flip-back, 3D NOESY-TROSY and TOCSY-TROSY experiments

Previously we demonstrated a sensitivity enhancement of the original TROSY experiment by a factor of by the use of the sensitivity enhanced TROSY (en-TROSY) scheme. Here, we develop a gradient and sensitivity enhanced TROSY experiment (gs-TROSY), which is designed to select magnetization transfer pathways that suppress spectral artifacts and reduce the number of required phase cycles while having minimal loss of sensitivity. Both of these experimental methods (en-TROSY and gs- TROSY) have been combined with a water flip-back scheme which provides a further increase in sensitivity for labile NH groups by avoiding water saturation. We also apply these TROSY schemes to 3D NOESY-TROSY and 3D TOCSY-TROSY experiments.     

Rhesus Monkey TRIM5α SPRY Domain Recognizes Multiple Epitopes That Span Several Capsid Monomers on the Surface of the HIV-1 Mature Viral Core

The restriction factor TRIM5α binds to the capsid protein of the retroviral core and blocks retroviral replication. The affinity of TRIM5α for the capsid is a major host tropism determinant of HIV and other primate immunodeficiency viruses, but the molecular interface involved in this host–pathogen interaction remains poorly characterized. Here we use NMR spectroscopy to investigate binding of the rhesus TRIM5α SPRY domain to a selection of HIV capsid constructs. The data are consistent with a model in which one SPRY domain interacts with more than one capsid monomer within the assembled retroviral core. The highly mobile SPRY v1 loop appears to span the gap between neighboring capsid hexamers making interhexamer contacts critical for restriction. The interaction interface is extensive, involves mobile loops and multiple epitopes, and lacks interaction hot spots. These properties, which may enhance resistance of TRIM5α to capsid mutations, result in relatively low affinity of the individual SPRY domains for the capsid, and the TRIM5α-mediated restriction depends on the avidity effect arising from the oligomerization of TRIM5α.     

Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY

This paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the water flip back technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.     

Structural basis of the transition-state stabilization in antibody-catalyzed hydrolysis

The catalytic antibody 6D9, which was raised against a transition-state analogue (TSA), catalyzes the hydrolysis of a non-bioactive chloramphenicol monoester to generate chloramphenicol. It has been shown that 6D9 utilizes the binding affinity in the catalysis; the differential affinity of the TSA relative to the substrate is equal to the rate enhancement. To reveal the recognition mechanism of 6D9 for the TSA and the substrate, we performed NMR analysis of the Fv fragment of 6D9 (6D9-Fv), together with site-directed mutagenesis and stopped-flow kinetic analyses. Among six 6D9-Fv mutants, Y58(H)A and W100i(H)A displayed significant reductions in their affinities to the TSA, while their substrate-binding affinities were identical with that of the wild-type 6D9-Fv. The stopped-flow kinetic studies revealed that the TSA binding to 6D9-Fv occurred by an induced-fit mechanism. In contrast, no induced-fit type of TSA-binding mechanism was observed for Y58(H)A and W100i(H)A. From NMR experiments, we identified the residues with chemical shifts that were perturbed by the ligand-binding. The residues affected by the TSA binding were located on the TSA-binding site determined by the X-ray study, and on the regions far from the binding site. On the other hand, the residues affected by the substrate binding were localized on the TSA-binding site. As for W100i(H)A, no residue other than those in the binding site was affected by the ligand binding. On the basis of these results and the crystal structure, we concluded that the TSA binding induced a conformational change involving the formation of aromatic-aromatic interactions and a hydrogen bond. These interactions can account for the differential affinity for the TSA relative to the substrate. W100i(H) probably plays an important role in inducing the conformational changes. The present NMR studies have enabled us to visualize the concept of transition-state stabilization in enzymatic catalysis, in which the transition-state contacts are better than those of the substrate.     

Transverse relaxation optimized HCN experiment for nucleic acids: Combining the advantages of TROSY and MQ spin evolution

A three-dimensional MQ-TROSY-HCN pulse sequence is presented which provides intra-base and sugar-to-base correlations for ¹³C, ¹⁵N labeled nucleic acids (RNA, DNA). The experiment simultaneously exploits the favorable relaxation properties of ¹H-¹³C multiple quantum coherence for sugar carbons and of ¹³C TROSY-type spin evolution for base carbons. MQ-TROSY-HCN thus combines the advantages of MQ-HCN for sugar-to-base and TROSY-HCN for intra-base correlations in a single experiment. In addition, two slightly different implementations of the MQ-TROSY-HCN experiment ensure optimal performance for small and larger oligonucleotides, respectively. The advantages of the MQ-TROSY-HCN experiment compared to the best previous implementations of HCN are demonstrated for a 33 nucleotide RNA aptamer.     

Cross-relaxation bottleneck in water-lysozyme proton magnetization exchange

The proton spin-lattice relaxation parameters in natural and deuterated lysozyme solutions have been measured as a function of temperature (0-50 degrees C). The variation of the apparent magnitudes of the water proton magnetizations in the solutions with temperature indicates that magnetic coupling mixes protein and water proton magnetizations. The results are consistent with an exchange cross-relaxation model (Hills, B. P., Mol Phys 1992, 76, 489-508) in which the cross-relaxation acts between the labile and nonlabile protons, rather than between water and protein protons. Although this cross-relaxation pathway clearly affects the observed magnetization fractions in this protein solution, its influence on the relaxation rates is less apparent.     

Direct determination of the heteronuclear T1/T2 ratio by off-resonance steady-state magnetization measurement: Investigation of the possible application to fast exchange characterization of 15N-labeled proteins

The ¹⁵N steady-state magnetization in the presence of off-resonance rf irradiation is an analytical function of the T₁/T₂ ratio and of the angle between the ¹⁵N effective field axis and the static magnetic field direction. This relation holds whatever the relaxation mechanisms due to motions on the nanosecond time scale, and the size of the spin system. If motions on the micro- to millisecond time scale are present (fast exchange), the same observable depends also on their spectral density at the frequency of the effective field. The cross-peak intensity in each 2D ¹⁵N-¹H correlation map is directly related to the dynamic parameters, so that the characterization of fast exchange phenomena by this method is in principle less time-consuming than the separate measurement of self-relaxation rates. The theory of this approach is described. Its practical validity is experimentally evaluated on a ¹⁵N-labeled 61 amino acid neurotoxin. It turns out that existing equipments lead to non-negligible biases. Their consequences for the accuracy attainable, at present, by this method are investigated in detail.     

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between ¹H^N-¹³C, ¹⁵N-¹³C, ¹H^N-¹³C _i–1, ¹⁵N-¹³C _i–1, ¹H^N-¹³C_i–1, ¹⁵N-¹³C_i–1, and ¹³C_i–1-¹³C _i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional ¹⁵N-¹H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.     

Study of the water-collagen system by NMR cross relaxation experiments

The present work is a study of cross relaxation between water and collagen by NMR pulse techniques. This cross relaxation experiments is easy to set up for NMR spectrometers and gives further information supplementary to other experiments. Relaxation parameters derived from this method provide a sensitive means of monitoring the hydration-dependent behaviour of different types of collagen.     

Suppression of anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme

A simple modification of the TROSY pulse transfer scheme, suggested by Yang and Kay [J. Biomol. NMR 13 (1999) 3–10], is proposed which results in the suppression of unwanted anti-TROSY lines without any extra loss in sensitivity. The higher sensitivity of this TROSY transfer scheme therefore becomes available for 2D [¹⁵N, ¹H] TROSY correlation and 3D/4D ¹⁵N separated NOESY type experiments where complete suppression of the broad anti-TROSY lines is essential.     

Structure of the LSm657 complex: an assembly intermediate of the LSm1-7 and LSm2-8 rings

The nuclear LSm2-8 (like Sm) complex and the cytoplasmic LSm1-7 complex play a central role in mRNA splicing and degradation, respectively. The LSm proteins are related to the spliceosomal Sm proteins that form a heteroheptameric ring around small nuclear RNA. The assembly process of the heptameric Sm complex is well established and involves several smaller Sm assembly intermediates. The assembly of the LSm complex, however, is less well studied. Here, we solved the 2.5 Å-resolution structure of the LSm assembly intermediate that contains LSm5, LSm6, and LSm7. The three monomers display the canonical Sm fold and arrange into a hexameric LSm657-657 ring. We show that the order of the LSm proteins within the ring is consistent with the order of the related SmE, SmF, and SmG proteins in the heptameric Sm ring. Nonetheless, differences in RNA binding pockets prevent the prediction of the nucleotide binding preferences of the LSm complexes. Using high-resolution NMR spectroscopy, we confirm that LSm5, LSm6, and LSm7 also assemble into a  60-kDa hexameric ring in solution. With a combination of pull-down and NMR experiments, we show that the LSm657 complex can incorporate LSm23 in order to assemble further towards native LSm rings. Interestingly, we find that the NMR spectra of the LSm57, LSm657-657, and LSm23-657 complexes differ significantly, suggesting that the angles between the LSm building blocks change depending on the ring size of the complex. In summary, our results identify LSm657 as a plastic and functional building block on the assembly route towards the LSm1-7 and LSm2-8 complexes.     

A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands

Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe a strategy for resonance assignment in selectively labeled proteins. Our approach involves a spin-labeled analog of a ligand of which the three-dimensional structure in complex with the target protein is known. Other methods for introduction of the spin label are possible. The paramagnetic center causes faster relaxation of all neighboring nuclei in a distance-dependent manner. Measurement of this effect allows to deduce distances between isotopically labeled residues and the paramagnetic center which can be used for resonance assignment. The method is demonstrated for the catalytic domain of Abl kinase in complex with the inhibitor, STI571.     

Structural polymorphism of chromodomains in Chd1

Chromodomain from heterochromatin protein 1 and polycomb protein is known to be a lysine-methylated histone H3 tail-binding module. Chromo-helicase/ATPase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodeling factor, containing two tandem chromodomains. In human CHD1, both chromodomains are essential for specific binding to a K4 methylated histone H3 (H3 MeK4) peptide and are found to bind cooperatively in the crystal structure. For the budding yeast homologue, Chd1, the second but not the first chromodomain was once reported to bind to an H3 MeK4 peptide. Here, we reveal that neither the second chromodomain nor a region containing tandem chromodomains from yeast Chd1 bind to any lysine-methylated or arginine-methylated histone peptides that we examined. In addition, we examined the structures of the chromodomains from Chd1 by NMR. Although the tertiary structure of the region containing tandem chromodomains could not be obtained, the secondary structure deduced from NMR is well conserved in the tertiary structures of the corresponding first and second chromodomains determined individually by NMR. Both chromodomains of Chd1 demonstrate a structure similar to that of the corresponding part of CHD1, consisting of a three-stranded beta-sheet followed by a C-terminal alpha-helix. However, an additional helix between the first and second beta-strands, which is found in both of the first chromodomains of Chd1 and CHD1, is positioned in an entirely different manner in Chd1 and CHD1. In human CHD1 this helix forms the peptide-binding site. The amino acid sequences of the chromodomains could be well aligned on the basis of these structures. The alignment showed that yeast Chd1 lacks several key functional residues, which are responsible for specific binding to a methylated lysine residue in other chromodomains. Chd1 is likely to have no binding affinity for any H3 MeK peptide, as found in other chromodomain proteins.