Angular dependence of dipole-dipole-Curie-spin cross-correlation effects in high-spin and low-spin paramagnetic myoglobin

The ¹⁵N-HSQC spectra of low-spin cyano-met-myoglobin and high-spin fluoro-met-myoglobin were assigned and dipole-dipole-Curie-spin cross-correlated relaxation rates measured. These cross-correlation rates originating from the dipolar ¹H-¹⁵N interaction and the dipolar interaction between the ¹H and the Curie spin of the paramagnetic center contain long-range angular information about the orientation of the ¹H-¹⁵N bond with respect to the iron-¹H vector, with information measurable up to 11 Å from the metal for the low-spin complex, and between 10 to 25 Å for the high-spin complex. Comparison of the experimental data with predictions from crystal structure data showed that the anisotropy of the magnetic susceptibility tensor in low spin cyano-met-myoglobin significantly influences the cross-correlated dipole-dipole-Curie-spin relaxation rates.     

A novel experiment for the quantitative measurement of CSA(1HN)/CSA(15N) cross-correlated relaxation in 15N-labeled proteins*

An experiment is presented which allows for the quantitative measurement of the relaxation interference between the ¹H_N CSA and ¹⁵N CSA interactions in ¹⁵N labeled proteins. A constant-time buildup scheme is used to measure the differential relaxation rate, , between double-quantum (DQ) and zero-quantum (ZQ) ¹H_N-¹⁵N coherences. The CSA/CSA experiment was recorded at three different B_o field strengths. The CSA(¹H_N)/CSA(¹⁵N) cross-correlation rate was obtained from the linear fit of the measured rate, , versus B_o ² for 77 residues of the EH2 domain from mouse Eps15.     

Comparison of fast backbone dynamics at amide nitrogen and carbonyl sites in dematin headpiece C-terminal domain and its S74E mutant

We perform a detailed comparison of fast backbone dynamics probed at amide nitrogen versus carbonyl carbon sites for dematin headpiece C-terminal domain (DHP) and its S74E mutant (DHPS74E). Carbonyl dynamics is probed via auto-correlated longitudinal rates and transverse C′/C′-C^α CSA/dipolar and C′/C′–N CSA/dipolar cross-correlated rates, while ¹⁵N data are taken from a previous study. Resulting values of effective order parameters and internal correlation times support the conclusion that C′ relaxation reports on a different subset of fast motions compared to those probed at N–H bond vectors in the same peptide planes. ¹³C′ order parameters are on the average 0.08 lower than ¹⁵N order parameters with the exception of the flexible loop region in DHP. The reduction of mobility in the loop region upon the S74E mutation can be seen from the ¹⁵N order parameters but not from the ¹³C order parameters. Internal correlation times at ¹³C′ sites are on the average an order of magnitude longer than those at ¹⁵N sites for the well-structured C-terminal subdomains, while the more flexible N-terminal subdomains have more comparable average internal correlation times.     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Temperature dependence of fast carbonyl backbone dynamics in chicken villin headpiece subdomain

Temperature-dependence of protein dynamics can provide information on details of the free energy landscape by probing the characteristics of the potential responsible for the fluctuations. We have investigated the temperature-dependence of picosecond to nanosecond backbone dynamics at carbonyl carbon sites in chicken villin headpiece subdomain protein using a combination of three NMR relaxation rates: ¹³C′ longitudinal rate, and two cross-correlated rates involving dipolar and chemical shift anisotropy (CSA) relaxation mechanisms, ¹³C′/¹³C′-¹³C^α CSA/dipolar and ¹³C′/¹³C′–¹⁵N CSA/dipolar. Order parameters have been extracted using the Lipari-Szabo model-free approach assuming a separation of the time scales of internal and molecular motions in the 2–16°C temperature range. There is a gradual deviation from this assumption from lower to higher temperatures, such that above 16°C the separation of the time scales is inconsistent with the experimental data and, thus, the Lipari-Szabo formalism can not be applied. While there are variations among the residues, on the average the order parameters indicate a markedly steeper temperature dependence at backbone carbonyl carbons compared to that probed at amide nitrogens in an earlier study. This strongly advocates for probing sites other than amide nitrogen for accurate characterization of the potential and other thermodynamics characteristics of protein backbone.     

Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation

The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei ¹³C, ¹H,¹³CO, ¹⁵N and ¹H^N can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state ¹³CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between ¹H(i)-¹³C(i)/¹⁵N(i)-¹H^N(i) and ¹H(i–1)-¹³C(i–1)/¹⁵N(i)-¹H^N(i) dipole-dipole mechanisms in ¹⁵N,¹³C-labeled proteins. The approach is illustrated with an application to ¹³C,¹⁵N-labeled ubiquitin.     

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly <Superscript>15</Superscript>N labeled integral membrane proteins in magnetically aligned lipid bilayers

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring ¹H-¹⁵N dipolar couplings (DC) and ¹⁵N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([¹H,¹⁵N]-SE-PISEMA-PDSD). The incorporation of 2D ¹⁵N/¹⁵N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the ¹⁵N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.     

REDOR: an assessment of the efficacy of dipolar recoupling with adiabatic inversion pulses

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3–6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model ¹⁵N-¹³C spin systems, (¹³C,¹⁵N) Aib-(¹⁵N) Aib-NH₂ (Aib = -aminoisobutyric acid) and (1-¹³C,¹⁵N) glycine.     

Quantitative estimation of magnitude and orientation of the CSA tensor from field dependence of longitudinal NMR relaxation rates

A method is presented that makes it possible to estimate both the orientation and the magnitude of the chemical shift anisotropy (CSA) tensor in molecules with a pair of spin 1/2 nuclei, typically ¹³C-¹H or ¹⁵ N-¹H. The method relies on the fact that the longitudinal cross-correlation rate as well as a linear combination of the autorelaxation rates of longitudinal heterospin magnetization, longitudinal two-spin order and longitudinal proton magnetization are proportional to the spectral density at the Larmor frequency of the heterospin. Therefore the ratio between the cross-correlation rate and the above linear combination is independent of the dynamics. From the field dependence of the ratio both the magnitude and the orientation of the CSA tensor can be estimated. The method is applicable to molecules in all motional regimes and is not limited to molecules in extreme narrowing or slow tumbling, nor is it sensitive to chemical exchange broadening. It is tested on the 22 amino acid residue peptide motilin, selectively ¹³ C labeled in the ortho positions in the ring of the single tyrosine residue. In the approximation of an axially symmetric ¹³C CSA tensor, the symmetry axis of the CSA tensor makes an angle of 23° ± 1° to the ¹³ C-¹H bond vector, and has a magnitude of 156 ± 5 ppm. This is in close agreement with solid-state NMR data on tyrosine powder [Frydman et al. (1992) Isr. J. Chem., 32, 161–164].     

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.     

Can PISEMA experiments be used to extract structural parameters for mobile β-barrels?

The effect of mobility on ¹⁵N chemical shift/¹⁵N–¹H dipolar coupling (PISEMA) solid state NMR experiments applied to macroscopically oriented β-barrels is assessed using molecular dynamics simulation data of the NalP autotransporter domain embedded in a DMPC bilayer. In agreement with previous findings for α-helices, the fast librational motion of the peptide planes is found to have a considerable effect on the calculated PISEMA spectra. In addition, the dependence of the chemical shift anisotropy (CSA) and dipolar coupling parameters on the calculated spectra is evaluated specifically for the β-barrel case. It is found that the precise choice of the value of the CSA parameters σ₁₁,σ₂₂ and σ₃₃ has only a minor effect, whereas the choice of the CSA parameter θ shifts the position of the peaks by up to 20 ppm and changes the overall shape of the spectrum significantly. As was found for α-helices, the choice of the NH bond distance has a large effect on the dipolar coupling constant used for the calculations. Overall, it is found that the alternating β-strands in the barrel occupy distinct regions of the PISEMA spectra, forming patterns which may prove useful in peak assignment.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-5094-5     

Spin-state selection for increased confidence in cross-correlation rates measurements

A new approach is described for measuring chemical shift anisotropy (CSA)/dipolar cross-correlated relaxation (CCR) rates based on the selection of the individual ¹⁵N doublet components prior to the relaxation period. The method uses the spin-state-selective element (S³E) of Sørensen and co-authors [Meissner et al. (1997) J. Mag. Reson., 128, 92–97]. The main advantage of the new method compared to other J-resolved experiments is that it does not create problems of additional signal overlap encountered in coupled spectra. At the same time, this approach allows a simpler control of magnetization pathways than the indirect methods. The method is demonstrated for the B3 domain of protein G.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7562-8     

Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy

We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include ¹⁵N T ₁ relaxation times measured at two different magnetic fields as well as ¹H–¹⁵N dipole, ¹⁵N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T ₂ in the solid-state. In addition, global order parameters are included from a ¹H,¹⁵N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore–Lipari–Szabo theory. We find slow motional correlation times in the range of 5 and 150 ns. Assuming a wobbling in a cone motion, the amplitude of motion of the respective amide moiety is on the order of 10° for the half-opening angle of the cone in most of the cases. The experiments are demonstrated using a perdeuterated sample of the chicken α-spectrin SH3 domain.     

Rapid automated determination of chemical shift anisotropy values in the carbonyl and carboxyl groups of fd-y21m bacteriophage using solid state NMR

Determination of chemical shift anisotropy (CSA) in immobilized proteins and protein assemblies is one of several tools to determine protein dynamics on the timescales of microseconds and faster. The large CSA values of C=O groups in the rigid limit makes them in particular attractive for measurements of large amplitude motions, or their absence. In this study, we implement a 3D R-symmetry-based sequence that recouples the second spatial component of the ¹³C CSA with the corresponding isotropic ¹³C′–¹³C cross-peaks in order to probe backbone and sidechain dynamics in an intact fd-y21m filamentous phage viral capsid. The assignment of the isotropic cross-peaks and the analysis were conducted automatically using a new software named ‘Raven’. The software can be utilized to auto-assign any 2D ¹³C–¹³C or ¹⁵N–¹³C spectrum given a previously-determined assignment table and generates simultaneously all intensity curves acquired in the third dimension. Here, all CSA spectra were automatically generated, and subsequently matched against a simulated set of CSA curves to yield their values. For the multi-copy, 50-residue-long protein capsid of fd-y21m, the backbone of the helical region is rigid, with reduced CSA values of ~?12.5 kHz (~?83 ppm). The N-terminus shows motionally-averaged CSA lineshapes and the carboxylic sidechain groups of four residues indicate large amplitude motions for D4, D5, D12 and E20. The current results further strengthen our previous studies of ¹⁵N CSA values and are in agreement with qualitative analysis of ¹³C–¹³C dipolar build-up curves, which were automatically obtained using our software. Our automated analysis technique is general and can be applied to study protein structure and dynamics, with data resulting from experiments that probe different variables such as relaxation rates and scaled anisotropic interactions.

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Determination of the residue-specific 15N CSA tensor principal components using multiple alignment media

The individual components of the backbone ¹⁵N CSA tensor, σ₁₁, σ₂₂, σ₃₃, and the orientation of σ₁₁ relative to the NH bond described by the angle β have been determined for uniformly labeled ¹⁵N, ¹³C ubiquitin from partial alignment in phospholipid bicelles, Pf1 phage, and poly(ethylene glycol) by measuring the residue-specific residual dipolar couplings and chemical shift deviations. No strong correlation between any of the CSA tensor components is observed with any single structural feature. However, the experimentally determined tensor components agree with the previously determined average CSA principal components [Cornilescu and Bax (2000) J. Am. Chem. Soc. 122, 10143–10154]. Significant deviations from the averages coincide with residues in β-strand or extended regions, while α-helical residue tensor components cluster close to the average values.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Intraresidue 1H-15N-13C′ and 1Hα-13Cα-13C′ dipole-CSA relaxation interference as a source of constraints for structural refinement of metal-binding sites in zinc-finger proteins

¹H(i)-¹⁵N(i)-¹³C(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the ¹³C,¹⁵N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane torsion angles and taken from the solution structure. ¹H(i)-¹⁵N(i)-¹³C(i) as well as ¹³C(i)-¹H(i)-¹³C(i) dipole-CSA cross-correlation rates were subsequently used to improve the geometry of the metal binding site. The optimized dihedral angles of the two zinc-binding sites in qCRP2(LIM2) are in better agreement with values obtained from crystal structures of other zinc-finger proteins and thus establish the utility of this approach to improve the metal-binding site geometry of zinc-finger proteins studied by NMR spectroscopy in solution.     

Quantifying Lipari–Szabo modelfree parameters from 13CO NMR relaxation experiments

It is proposed to obtain effective Lipari–Szabo order parameters and local correlation times for relaxation vectors of protein ¹³CO nuclei by carrying out a ¹³CO-R₁ auto relaxation experiment, a transverse CSA/dipolar cross correlation and a transverse ¹³CO CSA/¹³CO–¹⁵N CSA/dipolar cross correlation experiment. Given the global rotational correlation time from ¹⁵N relaxation experiments, a new program COMFORD (CO-Modelfree Fitting Of Relaxation Data) is presented to fit the ¹³CO data to an effective order parameter , an effective local correlation time and the orientation of the CSA tensor with respect to the molecular frame. It is shown that the effective is least sensitive to rotational fluctuations about an imaginary axis and most sensitive to rotational fluctuations about an imaginary axis parallel to the NH bond direction. As such, the information is fully complementary to the ¹⁵N relaxation order parameter, which is least sensitive to fluctuations about the NH axis and most sensitive to fluctuations about the axis. The new paradigm is applied on data of Ca²⁺ saturated Calmodulin, and on available literature data for Ubiquitin. Our data indicate that the order parameters rapport on slower, and sometimes different, motions than the ¹⁵N relaxation order parameters. The CO local correlation times correlate well with the calmodulin’s secondary structure. Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

The influence of internuclear spatial distribution and instrument noise on the precision of distances determined by solid state NMR of isotopically enriched proteins

The relative merits of different isotopic enrichment strategies that might be used in solid state NMR protein structure determinations are explored. The basis for comparison of these merits is the determination of the relative uncertainties in rates measured by a generalized dipolar recoupling experiment. The different schemes considered use ¹³C, ¹⁵N and ²H labeling of ubiquitin with homonuclear magnetization-transfer type experiments under magic-angle spinning (MAS). Specific attention is given to the sensitivity of the predicted relative precisions to variation in natural nuclear density distribution and noise levels. A framework is suggested to gauge the precision of measurement of a given dipolar coupling constant, and the potential for a set of such measurements to constrain structure calculations is explored. The distribution of nuclei in homonuclear ¹⁵N and ¹H dipolar recoupling spin-exchange experiments appear to provide the most promising tertiary structure information for uniformly labeled ubiquitin.     

Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using ¹⁵N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R ₂ values in the -helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T ₁ values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R ₂/R ₁ is anisotropic (D _par/D _perp=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with ¹⁵N relaxation rates and the estimation of R _ex terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.     

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.