Multiple alignment tensors from a denatured protein

The structural content of the denatured state has yet to be fully characterized. In recent years, large residual dipolar couplings (RDCs) from denatured proteins have been observed under alignment conditions produced by bicelles and strained polyacrylamide gels. In this report, we describe efforts to extend our picture of the residual structure in denatured nuclease by measuring RDCs with multiple alignment tensors. Backbone amide 15N-1H RDCs were collected from 4 M urea for a total of eight RDC data sets. The RDCs were analyzed by singular value decomposition (SVD) to determine the number of independent alignment tensors present in the data. On the basis of the resultant singular values and propagated error estimates, it is clear that there are at least three independent alignment tensors. These three independent RDC datasets can be reconstituted as orthogonal linear combinations, (OLC)-RDC datasets, of the eight actually recorded. The first, second, and third OLC-RDC datasets are highly robust to the removal of any single experimental RDC dataset, establishing the presence of three independent alignment tensors, sampled well above the level of experimental uncertainty. The observation that the RDC data span three or more dimensions of the five-dimensional parameter space demonstrates that the ensemble average structure of denatured nuclease must be asymmetric with respect to these three orthogonal principal axes, which is not inconsistent with earlier work demonstrating that it has a nativelike topology.     

De novo determination of protein backbone structure from residual dipolar couplings using Rosetta

As genome-sequencing projects rapidly increase the database of protein sequences, the gap between known sequences and known structures continues to grow exponentially, increasing the demand to accelerate structure determination methods. Residual dipolar couplings (RDCs) are an attractive source of experimental restraints for NMR structure determination, particularly rapid, high-throughput methods, because they yield both local and long-range orientational information and can be easily measured and assigned once the backbone resonances of a protein have been assigned. While very extensive RDC data sets have been used to determine the structure of ubiquitin, it is unclear to what extent such methods will generalize to larger proteins with less complete data sets. Here we incorporate experimental RDC restraints into Rosetta, an ab initio structure prediction method, and demonstrate that the combined algorithm provides a general method for de novo determination of a variety of protein folds from RDC data. Backbone structures for multiple proteins up to approximately 125 residues in length and spanning a range of topological complexities are rapidly and reproducibly generated using data sets that are insufficient in isolation to uniquely determine the protein fold de novo, although ambiguities and errors are observed for proteins with symmetry about an axis of the alignment tensor. The models generated are not high-resolution structures completely defined by experimental data but are sufficiently accurate to accelerate traditional high-resolution NMR structure determination and provide structure-based functional insights.     

Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.     

Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins

Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.     

Periodicity in residual dipolar couplings and nucleic acid structures

The periodicity in nucleic acid duplex structures is shown to be correlated to the periodicity in residual dipolar couplings (RDCs) in the form of an "RDC wave". This "RDC wave" is characteristic of the alignment of the duplex in the magnetic field, and hence fitting of the data allows the duplex global orientation (, Phi) to be extracted. Further, because the "RDC wave" is fit as a data set of a corresponding secondary structure element, the degeneracy problem is greatly reduced. Consequently, with the global orientation (, Phi) determined, local bond vector conformations are defined. The fit is demonstrated in the examples of the imino RDCs of the negative regulator of splicing RNA fragment (NRS23) and for the C1'H1' RDCs of the Dickerson dodecamer.     

Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins

High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR. Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins. (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E. coli sulfite reductase. These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame. The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample. To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model. Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.     

Composite alignment media for the measurement of independent sets of NMR residual dipolar couplings

The measurement of independent sets of NMR residual dipolar couplings (RDCs) in multiple alignment media can provide a detailed view of biomolecular structure and dynamics, yet remains experimentally challenging. It is demonstrated here that independent sets of RDCs can be measured for ubiquitin using just a single alignment medium composed of aligned bacteriophage Pf1 particles embedded in a strained polyacrylamide gel matrix. Using this composite medium, molecular alignment can be modulated by varying the angle between the directors of ordering for the Pf1 and strained gel matrix, or by varying the ionic strength or concentration of the Pf1 particles. This approach offers significant advantages in that greater experimental control can be exercised over the acquisition of multi-alignment RDC data while a homogeneous chemical environment is maintained across all of the measured RDC data.     

Construction of covalent membrane protein complexes and high-throughput selection of membrane mimics

The association of transmembrane (TM) helices underlies membrane protein structure and folding. Structural studies of TM complexes are limited by complex stability and the often time-consuming selection of suitable membrane mimics. Here, methodology for the efficient, preparative scale construction of covalent TM complexes and the concomitant high-throughput selection of membrane mimics is introduced. For the employed integrin αIIbβ3 model system, the methodology identified phospholipid bicelles, including their specific composition, as the best membrane mimic. The method facilitates structure determination by NMR spectroscopy as exemplified by the measurement of previously inaccessible residual dipolar couplings and (15)N relaxation parameters.     

An Alignment Medium for Measuring Residual Dipolar Couplings in Pure DMSO: Liquid Crystals from Graphene Oxide Grafted with Polymer Brushes

Residual dipolar couplings (RDCs) have attracted attention in light of their great impact on the structural elucidation of organic molecules. However, the effectiveness of RDC measurements is limited by the shortage of alignment media compatible with widely used organic solvents, such as DMSO. Herein, we present the first liquid crystal (LC) based alignment medium that is compatible with pure DMSO, thus enabling RDC measurements of polar and intermediate polarity molecules. The liquid crystals were obtained by grafting polymer brushes onto graphene oxide (GO) using free radical polymerization. The resulting new medium offers several advantages, such as absence of background signals, narrow line shapes, and tunable alignment. Importantly, this medium is compatible with π‐conjugated molecules. Moreover, sonication‐induced fragmentation can reduce the size of GO sheets. The resulting anisotropic medium has moderate alignment strength, which is a prerequisite for an accurate RDC measurement.     

Long‐Range Residual Dipolar Couplings: A Tool for Determining the Configuration of Small Molecules

Together with NOE and J coupling, one‐bond residual dipolar coupling (RDC), which reports on the three‐dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few C? H bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long‐range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one‐bond RDCs. Herein we present a method for extracting the long‐range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non‐protonated carbon atoms. Alignment tensors fitted directly to the total long‐range couplings (T=J+D) enabled straightforward analysis of both the long‐range and one‐bond RDCs for strychnine.     

Magnetically aligned phospholipid bilayers with large q ratios stabilize magnetic alignment with high order in the gel and L(alpha) phases

The magnetic alignment behavior ofbicelles (magnetically alignable phospholipid bilayered membranes) as a function of the q ratio (1,2-dihexanoyl-sn-glycerol phosphatidylcholine/1,2-dimyristoyl-sn-glycerol phosphatidylcholine mole ratio) and temperature was studied by spin-labeled X-band electron paramagnetic resonance (EPR) spectroscopy and solid-state 2H and 31P NMR spectroscopy. Well-aligned bicelle samples were obtained at 45 degrees C for q ratios between 2.5 and 9.5 in both the EPR and NMR spectroscopic studies. The molecular order of the system, S(mol), increased as the q ratio increased and as the temperature decreased. For higher q ratios (> or = 5.5), bicelles maintained magnetic alignment when cooled below the main phase transition temperature (approximately 30 degrees C when in the presence of lanthanide cations), which is the first time, to our knowledge, that bicelles were magnetically aligned in the gel phase. For the 9.5 q ratio sample at 25 degrees C, S(mol) was calculated to be 0.83 (from 2H NMR spectra, utilizing the isotopic label perdeuterated 1,2-dimyristoyl-sn-glycerol phosphatidylcholine) and 0.911 (from EPR spectra utilizing the spin probe 3beta-doxyl-5alpha-cholestane). The molecular ordering of the high q ratio bicelles is comparable to literature values of S(mol) for both multilamellar vesicles and macroscopically aligned phospholipid bilayers on glass plates. The order parameter S(bicelle) revealed that the greatest degree of bicelle alignment was found at higher temperatures and larger q ratios (S(bicelle) = -0.92 for q ratio 8.5 at 50 degrees C).     

Increasing the accuracy of solution NMR structures of membrane proteins by application of residual dipolar couplings. High-resolution structure of outer membrane protein A

The structure determination of membrane proteins is one of the most challenging applications of solution NMR spectroscopy. The paucity of distance information available from the highly deuterated proteins employed requires new approaches in structure determination. Here we demonstrate that significant improvement in the structure accuracy of the membrane protein OmpA can be achieved by refinement with residual dipolar couplings (RDCs). The application of charged polyacrylamide gels allowed us to obtain two alignments and accurately measure numerous heteronuclear dipolar couplings. Furthermore, we have demonstrated that using a large set of RDCs in the refinement can yield a structure with 1 A rms deviation to the backbone of the high-resolution crystal structure. Our simulations with various data sets indicate that dipolar couplings will be critical for obtaining accurate structures of membrane proteins.     

The use of residual dipolar coupling for conformational analysis of structurally related natural products

Determining the conformational preferences of molecules in solution remains a considerable challenge. Recently, the use of residual dipolar coupling (RDC) analysis has emerged as a key method to address this. Whilst to date the majority of the applications have focused on biomolecules including proteins and DNA, the use of RDCs for studying small molecules is gaining popularity. Having said that, the method continues to develop, and here, we describe an early case study of the quantification of conformer populations in small molecules using RDC analysis. Having been inspired to study conformational preferences by unexpected differences in the NMR spectra and the reactivity of related natural products, we showed that the use of more established techniques was unsatisfactory in explaining the experimental observations. The use of RDCs provided an improved understanding that, following use of methods to quantify conformer populations using RDCs, culminated in a rationalisation of the contrasting diastereoselectivities observed in a ketone reduction reaction. Copyright © 2015 John Wiley & Sons, Ltd.     

NMR methods for studying the structure and dynamics of oncogenic and antihistaminic peptides in biomembranes

We present several applications of both wide-line and magic angle spinning (MAS) solid-state NMR of bicelles in which are embedded fragments of a tyrosine kinase receptor or enkephalins. The magnetically orientable bicelle membranes are shown to be of particular interest for studying the functional properties of lipids and proteins in a state that is very close to their natural environment. Quadrupolar, dipolar and chemical shielding interactions can be used to determine minute alterations of internal membrane dynamics and the orientation of peptides with respect to the membrane plane. MAS of bicelles can in turn lead to high-resolution proton spectra of hydrated membranes. Using deuterium-proton contrast methods one can then obtain pseudo-high-resolution proton spectra of peptides or proteins embedded in deuterated membranes and determine their atomic 3D structure using quasi-conventional liquid-state NMR methods.     

Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy

In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR.     

A high-resolution solid-state NMR approach for the structural studies of bicelles

Bicelles are increasingly being used as membrane mimicking systems in NMR experiments to investigate the structure of membrane proteins. In this study, we demonstrate the effectiveness of a 2D solid-state NMR approach that can be used to measure the structural constraints, such as heteronuclear dipolar couplings between 1H, 13C, and 31P nuclei, in bicelles without the need for isotopic enrichment. This method does not require a high radio frequency power unlike the presently used rotating-frame separated-local-field (SLF) techniques, such as PISEMA. In addition, multiple dipolar couplings can be measured accurately, and the presence of a strong dipolar coupling does not suppress the weak couplings. High-resolution spectra obtained from magnetically aligned DMPC:DHPC bicelles even in the presence of peptides suggest that this approach will be useful in understanding lipid-protein interactions that play a vital role in shaping up the function of membrane proteins.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

Magnetic Alignment of Polymer Macro‐Nanodiscs Enables Residual‐Dipolar‐Coupling‐Based High‐Resolution Structural Studies by NMR Spectroscopy

Experimentally measured residual dipolar couplings (RDCs) are highly valuable for atomic‐resolution structural and dynamic studies of molecular systems ranging from small molecules to large proteins by solution NMR spectroscopy. Here we demonstrate the first use of magnetic‐alignment behavior of lyotropic liquid‐crystalline polymer macro‐nanodiscs (>20 nm in diameter) as a novel alignment medium for the measurement of RDCs using high‐resolution NMR. The easy preparation of macro‐nanodiscs, their high stability against pH changes and the presence of divalent metal ions, and their high homogeneity make them an efficient tool to investigate a wide range of molecular systems including natural products, proteins, and RNA.     

Measurement of ribose carbon chemical shift tensors for A-form RNA by liquid crystal NMR spectroscopy

Incomplete motional averaging of chemical shift anisotropy upon weak alignment of nucleic acids and proteins in a magnetic field results in small changes in chemical shift. Knowledge of nucleus-specific chemical shift (CS) tensor magnitudes and orientations is necessary to take full advantage of these measurements in biomolecular structure determination. We report the determination by liquid crystal NMR of the CS tensors for all ribose carbons in A-form helical RNA, using a series of novel 3D NMR pulse sequences for accurate and resolved measurement of the ribose (13)C chemical shifts. The orientation of the riboses relative to the rhombic alignment tensor of the molecule studied, a stem-loop sequence corresponding to helix-35 of 23S rRNA, is known from an extensive set of residual dipolar couplings (RDC), previously used to refine its structure. Singular-value-decomposition fits of the chemical shift changes to this structure, or alternatively to a database of helical RNA X-ray structures, provide the CS tensor for each type of carbon. Quantum chemical calculations complement the experimental results and confirm that the most shielded tensor component lies approximately along the local carbon-oxygen bond axis in all cases and that shielding anisotropy for C3' and C4' is much larger than for C1' and C2', with C5' being intermediate.