Residual Dipolar Couplings In Proteins Research Articles

Flagella as a novel alignment medium for the measurement of residual dipolar couplings in proteins

The two flexible rod-like flagella (∼500 nm in diameter and 5-15 μm long) of Chlamydomonas reinhardtii, a unicellular green alga, can weakly align molecules in an external magnetic field, thereby enabling the measurement of various residual dipolar couplings in solution NMR spectroscopy.

Standard Tensorial Analysis of Local Ordering in Proteins from Residual Dipolar Couplings

Residual dipolar couplings (RDCs) in proteins arise from independent external medium-related and internal protein-related ordering of the spin-bearing probe. Griesinger et al. developed a method for treating RDCs in proteins. The global ordering is given in the standard manner by a rank 2 tensor specified in a known molecular frame, MF. The local ordering is described by the spherical harmonic ensemble averages, <Y(2m)(θ, φ)>, m = 0, ±1, ±2, also given in MF. From these quantities, a method we call mf-RDC derives the squared generalized order parameter (S(rdc)(2)), the amplitude (direction) of the anisotropic disorder, η (Φ′), and an approximation, (N−H)(eff), to the average probe orientation, i.e., to the local director. (N−H)(eff) is determined through a frame transformation where <Y(20)> is maximized. Φ′ is associated with a subsequent frame transformation where <Y(22) + Y(2−2)> is maximized. The mf-RDC method was applied previously to N−H and C−C(methyl) sites in ubiquitin. In this study, we convert the respective <Y(2m)(θ, φ)>'s into a Saupe tensor, which is diagonalized. This is the standard procedure. It yields the eigenvalues, S(xx), S(yy), and S(zz), and the Principal Axis System (PAS) of the rank 2 local ordering tensor, S(l). S(rdc)(2), η, and Φ′ can be recast as S(xx), S(yy), and S(zz). The mf-RDC frame transformations are not the same as the conventional Wigner rotation. The standard tensorial analysis provides new information. The contribution of local ordering rhombicity to S(rdc)(2) is evaluated. For the α-helix of ubiquitin, the main local ordering axis is assigned as C(i−1)(α) − C(i)(α); for the methyl sites, it is associated with the C−C(methyl) axis, as in mf-RDC. Ordering strength correlates with methyl type. The strength (rhombicity) of S(l) associated with picosecond−nanosecond local motions is reduced moderately (substantially) by nanosecond−millisecond local motions. A scheme for analyzing experimental RDCs based on the standard tensorial perspective, which allows for arbitrary orientation of the local director in the protein and of the PAS of S(l) in the probe, is formulated.

Single scan TROSY and E.COSY suite of experiments for the measurement of residual dipolar couplings in proteins

Simple modifications of the gradient enhanced version of the TROSY experiment allow to obtain four different spectra containing each one of the components of the H–N doublet in 15N labelled proteins. By measuring the difference in peak position among these four spectra, residual dipolar coupling values can be obtained for medium sized proteins. This experiment, which exploits the use of pulse field gradients to select the 15N coherence pathway, produces excellent results in terms of water suppression. Moreover, tuning of a single delay in the sequence reduces notably the presence of artifacts. The performance of this suite of experiments, that we called TEC (Trosy–E.Cosy) experiment, is tested against a J‐modulation method, inherently more accurate but more time consuming, for the accuracy in the observed values of residual dipolar couplings. For larger proteins, the use of this strategy allows to select the most favourable combination of peaks giving the sharpest signals.

A set of HA-detected experiments for measuring scalar and residual dipolar couplings

A new set of HCACO based three-dimensional NMR experiments for measuring residual dipolar couplings in proteins is presented. Using spin-state selection and editing in three dimensions, the experiments allow accurate measurement of intraresidual (1)D(C'H(alpha)), (1)D(C'C(alpha)) and (2)D(C'H(alpha)) scalar and residual dipolar couplings of (15) N/(13)C labeled proteins in D(2)O and dilute liquid crystals with minimal spectral crowding. The presented experiments are especially suitable for small or medium sized proline-rich proteins, or proteins that require high pH solvent conditions, making (1)H(N) detected experiments unattractive. In addition, the tetrahedral coordination of C(alpha) is superior to the planar peptide bond for determination of local alignments in partially structured polypeptides. For the efficient use of spectrometer time and to avoid complications arising from the varying magnitude of the alignment tensor during relatively long experiments, the (1)D(C'H(alpha)) and (2)D(C'H(alpha)) couplings can also be measured simultaneously in an E.COSY like manner with high accuracy. The pulse sequences are balanced for cross-correlation effects and minimized for relaxation losses. The pulse sequences are tested with a sample of (15)N/(13)C human ubiquitin. We find internuclear vector directions determined from the dipolar couplings to have an excellent correlation with those of ubiquitin's refined solution structure.

Measurement of scaled residual dipolar couplings in proteins using variable-angle sample spinning.

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.

Sensitivity-enhanced IPAP experiments for measuring one-bond 13C ′– 13C α and 13C α– 1H α residual dipolar couplings in proteins

Sensitivity-enhanced 2D IPAP experiments using the accordion principle for measuring one-bond 13C ′– 13C α and 1H α– 13C α dipolar couplings in proteins are presented. The resolution of the resulting spectra is identical to that of the decoupled HSQC spectra and the sensitivity of the corresponding 1D acquisitions are only slightly lower than those obtained with 3D HNCO and 3D HN(COCA)HA pulse sequences due to an additional delay 2 Δ. For cases of limited resolution in the 2D 15N– 1H N HSQC spectrum the current pulse sequences can easily be modified into 3D versions by introducing a poorly digitized third dimension, if so desired. The experiments described here are a valuable addition to the suites available for determination of residual dipolar couplings in biological systems.

Sensitivity-enhanced 2D IPAP, TROSY–anti-TROSY, and E.COSY experiments: alternatives for measuring dipolar [formula omitted] couplings

Sensitivity-enhanced versions of the IPAP, TROSY–anti-TROSY, and E.COSY experiments for measuring one-bond 15 N– 1 H N couplings are presented. Together with the previously developed sensitivity-enhanced E.COSY-type HSQC experiment they comprise a suite of sensitivity-enhanced experiments that allows one to chose the optimal spectrum for accurate measurement of one-bond 15 N– 1 H N residual dipolar couplings in proteins. Since one-bond 15 N– 1 H N residual dipolar couplings play uniquely important roles in structural NMR, these additional methods provide further tools for improving structure determination of proteins and other biological macromolecules.

Measurement of residual dipolar couplings from 1Halpha to 13Calpha and 15N using a simple HNCA-based experiment.

Novel NMR pulse schemes for simultaneous measurement of 1D(C)alpha(H)alpha and 2D(NH)alpha residual dipolar couplings in proteins is presented. We show that 2D(NH)alpha coupling can be very useful for protein structure determination. The 2D(NH)alpha coupling can be measured from 15N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large 1D(C)alpha(H)alpha coupling. The dynamic range of 2D(NH)alpha coupling is comparable to 1D(NC') coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on 15N/13C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both 1D(C)alpha(H)alpha and 2D(NH)alpha couplings, was obtained.

Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N– [formula omitted] and 15N– [formula omitted] and two-bond [formula omitted]– [formula omitted] residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N– 1 H N and 15N– 13 C ′ and two-bond 13 C ′ – 1 H N residual dipolar couplings in proteins. Compared with existing experiments, the ( δ, J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.

Residual dipolar couplings in protein NMR

AbstractPartial molecular alignment leads to an incomplete averaging of anisotropic magnetic interactions such as the magnetic dipole–dipole interaction, the chemical shift anisotropy, or the electric quadrupole interaction. The resulting so‐called “residual” anisotropic magnetic interactions currently become increasingly important in biomolecular NMR spectroscopy. In particular, residual dipolar couplings turn out to be extremely useful novel parameters for protein structure determination. The basic principles of this new methodology are discussed in the present review and recent applications are demonstrated. Different alignment mechanisms leading to partial orientation of protein molecules in solution are considered in detail. The influence of magnetic and electric fields applied to isotropic protein solutions is discussed. Various magnetically orienting media which can be admixed to protein solutions are described and compared. Experiments for the measurement of residual dipolar couplings in proteins and advantageous strategies for the use of residual dipolar couplings in structure calculations are reviewed. © 2001 John Wiley &amp; Sons, Inc. Concepts Magn Reson 13: 238–259, 2001

Pressure-stability of phospholipid bicelles: measurement of residual dipolar couplings under extreme conditions.

High-pressure NMR of proteins in solutions currently gains increasing interest. 3D structure determination of proteins under high pressure is, however, so far impossible due to the lack of NOE information. Residual dipolar couplings induced by the addition of magnetically orienting media are known to be capable of replacing NOE information to a very high extent. In the present contribution we study the pressure-stability of dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) bicelles and demonstrate the feasibility of measuring residual dipolar couplings in proteins under high pressure.

Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution.

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb3+)4 is effectively transmitted to the target protein (DHFR). 1H-15N HSQC IPAP experiments on the tagged complex containing 15N-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)4 allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to -7.4 Hz were measured at 600 MHz. Comparisons of 1H-15N HSQC spectra of 15N-DHFR-M13 alone and its complexes with CaM(Ca2+)4 and CaM(Tb3+)4 indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb3+) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).

Alignment of Biological Macromolecules in Novel Nonionic Liquid Crystalline Media for NMR Experiments

n-Alkyl-poly(ethylene glycol)/n-alkyl alcohol and glucopone/n-hexanol mixtures are shown to form dilute liquid crystalline phases in aqueous solution which are suitable for partial alignment of biological macromolecules in a magnetic field. The constituent compounds are commercially available and inexpensive. The poly(ethylene glycol)-based systems are uncharged and thus insensitive with respect to pH, little sensitive with respect to salt, and tolerant against high protein concentrations. They are demonstrated to be suitable for measurements of residual dipolar couplings in proteins, DNA, and a protein/DNA complex. The stability range of the lamellar phase with respect to temperature was explored for different mixtures and guidelines are provided for the use of these lyotropic systems in NMR spectroscopy.