Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.     

Principal component method for assessing structural heterogeneity across multiple alignment media

The recent availability of residual dipolar coupling measurements in a variety of different alignment media raises the question to what extent biomolecular structure and dynamics are differentially affected by their presence. A computational method is presented that allows the sensitive assessment of such changes using dipolar couplings measured in six or more alignment media. The method is based on a principal component analysis of the covariance matrix of the dipolar couplings. It does not require a priori structural or dynamic information nor knowledge of the alignment tensors and their orientations. In the absence of experimental errors, the covariance matrix has at most five nonzero eigenvalues if the structure and dynamics of the biomolecule is the same in all media. In contrast, differential structural and dynamic changes lead to additional nonzero eigenvalues. Characteristic features of the eigenvalue distribution in the absence and presence of noise are discussed using dipolar coupling data calculated from conformational ensembles taken from a molecular dynamics trajectory of native ubiquitin.     

Influence of the fluctuations of the alignment tensor on the analysis of the structure and dynamics of proteins using residual dipolar couplings

It has been suggested that the fluctuations of the alignment tensor can affect the results of procedures for characterizing the structure and the dynamics of proteins using residual dipolar couplings. We show here that the very significant fluctuations of the steric alignment tensor caused by the dynamics of proteins can be safely ignored when they do not correlate with those of the bond vectors. A detailed analysis of these correlations in the protein ubiquitin reveals that their effects are negligible for the analysis of backbone motions within secondary structure elements, but also that they may be significant in turns, loops and side chains, especially for bond vectors that have small residual dipolar couplings. Our results suggest that methods that explicitly consider the motions of the alignment tensor will be needed to study the large-scale structural fluctuations that take place on the millisecond timescale, which are often important for the biological function of proteins, from residual dipolar coupling measurements.     

NMR: prediction of molecular alignment from structure using the PALES software

Orientational restraints such as residual dipolar couplings promise to overcome many of the problems that traditionally limited liquid-state nuclear magnetic resonance spectroscopy. Recently, we developed methods to predict a molecular alignment tensor and thus residual dipolar couplings for a given molecular structure. This provides many new opportunities for the study of the structure and dynamics of proteins, nucleic acids, oligosaccharides and small molecules. This protocol details the use of the software PALES (Prediction of AlignmEnt from Structure) for prediction of an alignment tensor from a known three-dimensional (3D) coordinate file of a solute. The method is applicable to alignment of molecules in many neutral and charged orienting media and takes into account the molecular shape and 3D charge distribution of the molecule.     

Conformational dynamics and alignment properties of loop lanthanide-binding-tags (LBTs) studied in interleukin-1β

Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1β (Barthelmes et al. in J Am Chem Soc 133:808–819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1β constructs by measuring ¹⁵N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-τ_c-timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-τc dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.     

Line narrowing in spectra of proteins dissolved in a dilute liquid crystalline phase by band-selective adiabatic decoupling: Application to 1HN-15N residual dipolar coupling measurements

Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe ¹ H line broadening resulting from residual ¹ H-¹H dipolar couplings. In this communication we show that band-selective ¹H homonuclear decoupling during data acquisition is an efficient way to suppress residual ¹H-¹H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond ¹ H^N-¹⁵N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles.     

A Thorough Dynamic Interpretation of Residual Dipolar Couplings in Ubiquitin

The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822–5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098–6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cam

Residual dipolar couplings (RDCs) were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450(cam) (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The (1)D(NH) residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site and largely support previously reported differences between the open and closed states of CYP101A1.     

Evaluation of uncertainty in alignment tensors obtained from dipolar couplings

Residual dipolar couplings and their corresponding alignment tensors are useful for structural analysis of macromolecules. The error in an alignment tensor, derived from residual dipolar couplings on the basis of a known structure, is determined not only by the accuracy of the measured couplings but also by the uncertainty in the structure (structural noise). This dependence is evaluated quantitatively on the basis of simulated structures using Monte-Carlo type analyses. When large numbers of dipolar couplings are available, structural noise is found to result in a systematic underestimate of the magnitude of the alignment tensor. Particularly in cases where only few dipolar couplings are available, structural noise can cause significant errors in best-fitted alignment tensor values, making determination of the relative orientation of small fragments and evaluation of local backbone mobility from dipolar couplings difficult. An example for the protein ubiquitin demonstrates the inherent limitations in characterizing motions on the basis of local alignment tensor magnitudes.     

Submolecular unfolding units of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (¹D_CH) and long-range H-H residual dipolar couplings (ⁿD_HH) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the ⁿD_HH in natural abundance based on a ¹³C resolved ¹H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the ¹D_CH and the ⁿD_HH residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 16 glycosidic linkages.     

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.     

Calculation of z-coordinates and orientational restraints using a metal binding tag

We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings. Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.     

Induced alignment and measurement of dipolar couplings of an SH2 domain through direct binding with filamentous phage

Large residual ¹⁵N-¹H dipolar couplings have been measured in a Src homology II domain aligned at Pf1 bacteriophage concentrations an order of magnitude lower than used for induction of a similar degree of alignment of nucleic acids and highly acidic proteins. An increase in ¹ H and ¹⁵N protein linewidths and a decrease in T₂ and T₁ relaxation time constants implicates a binding interaction between the protein and phage as the mechanism of alignment. However, the associated increased linewidth does not preclude the accurate measurement of large dipolar couplings in the aligned protein. A good correlation is observed between measured dipolar couplings and predicted values based on the high resolution NMR structure of the SH2 domain. The observation of binding-induced protein alignment promises to broaden the scope of alignment techniques by extending their applicability to proteins that are able to interact weakly with the alignment medium.     

Novel Techniques for Weak Alignment of Proteins in Solution Using Chemical Tags Coordinating Lanthanide Ions

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.     

Quantitative conformational analysis of the core region of N-glycans using residual dipolar couplings, aqueous molecular dynamics, and steric alignment

A method is described for quantitatively investigating the dynamic conformation of small oligosaccharides containing an (16) linkage. It was applied to the oligosaccharide Man-(13) {Man- (16)}Man--O-Me, which is a core region frequently observed in N-linked glycans. The approach tests an aqueous molecular dynamics simulation, capable of predicting microscopic dynamics, against experimental residual dipolar couplings, by assuming that alignment is caused purely by steric hindrance. The experimental constraints were heteronuclear and homonuclear residual dipolar couplings, and in particular those within the (16) linkage itself. Powerful spin-state-selective pulse sequences and editing schemes were used to obtain the most relevant couplings for testing the model. Molecular dynamics simulations in water over a period of 50 ns were not able to predict the correct rotamer population at the (16) linkage to agree with the experimental data. However, this sampling problem could be corrected using a simple maximum likelihood optimisation, indicating that the simulation was modelling local dynamics correctly. The maximum likelihood prediction of the residual dipolar couplings was found to be an almost equal population of the gg and gt rotamer conformations at the (16) linkage, and the tg conformation was predicted to be unstable and unpopulated in aqueous solution. In this case all twelve measured residual dipolar couplings could be satisfied. This conformer population could also be used to make predictions of scalar couplings with the use of a previously derived empirical equation, and is qualitatively in agreement with previous predictions based on NMR, X-ray crystallography and optical data.     

Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field

For base-paired nucleic acids, variations in ¹ J _NH and the imino ¹H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the ¹ J _NH coupling for the imino proton then can be approximated by ¹ J _NH = (1.21Hz/ppm)δ_H − 103.5 ± 0.6 Hz, where δ_H represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (¹ J _NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA^Val and the alignment tensor determined from magnetic-field alignment of tRNA^Val agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work.     

Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the ²H NMR water signal and by the measurement of ¹H-¹⁵N residual dipolar couplings (RDC) in the archeal translation elongation factor 1. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.     

The utility of residual dipolar couplings in detecting motion in carbohydrates: application to sucrose

The solution structure and dynamics of sucrose are examined using a combination of NMR residual dipolar coupling and molecular mechanics force fields. It is found that the alignment tensors of the individual rings are different, and that fitting 35 measured residual dipolar couplings to structures with specific phi, psi values indicates the presence of three major conformations: phi, psi=(120 degrees ,270 degrees), (45 degrees, 300 degrees) and (90 degrees ,180 degrees). Furthermore, fitting two structures simultaneously to the 35 residual dipolar couplings results in a substantial improvement in the fits. The existence of multiple conformations having similar stabilities is a strong indication of motion, due to the interconversion among these states. Results from four molecular mechanics force fields are in general agreement with the experimental results. However, there are major disagreements between force fields. Because fits of residual dipolar couplings to structures are dependent on the force field used to calculate the structures, multiple force fields were used to interpret NMR data. It is demonstrated that the pucker of the fructofuranosyl ring affects the calculated potential energy surface, and the fit to the residual dipolar couplings data. Previously published 13C nuclear relaxation results suggesting that sucrose is rigid are not inconsistent with the present results when motional timescales are considered.