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.     

Direct prediction of residual dipolar couplings of small molecules in a stretched gel by stochastic molecular dynamics simulations

Residual dipolar couplings are highly useful NMR parameters for calculating and refining molecular structures, dynamics, and interactions. For some applications, however, it is inevitable that the preferred orientation of a molecule in an alignment medium is calculated a priori. Several methods have been developed to predict molecular orientations and residual dipolar couplings. Being beneficial for macromolecules and selected small‐molecule applications, such approaches lack sufficient accuracy for a large number of organic compounds for which the fine structure and eventually the flexibility of all involved molecules have to be considered or are limited to specific, well‐studied liquid crystals. We introduce a simplified model for detailed all‐atom molecular dynamics calculations with a polymer strand lined up along the principal axis as a new approach to simulate the preferred orientation of small to medium‐sized solutes in polymer‐based, gel‐type alignment media. As is shown by a first example of strychnine in a polystyrene/CDCl₃ gel, the simulations potentially enable the accurate prediction of residual dipolar couplings taking into account structural details and dynamic averaging effects of both the polymer and the solute. Copyright © 2015 John Wiley & Sons, Ltd.     

Connecting Discrete Stereoclusters by Using DFT and NMR Spectroscopy: The Case of Nivariol

The structural determination of small organic molecules is mainly undertaken by using NMR techniques, although it is increasingly supplemented by using computational methods. NMR parameters, such as chemical shifts and coupling constants, are extremely sensitive indicators of local molecular conformation and are a source of structural evidence. However, their interpretation is fairly challenging in many circumstances, such as the case of the new polyether squalene derivative nivariol, the structure of which was elucidated by means of NMR spectroscopy and DFT calculations. The potential flexibility of this molecule and the high number of quaternary carbon atoms that it contains make its configurational assignment very difficult. Moreover, the relative configuration of four separated stereoclusters was established and subsequently connected by using NOE and J‐based analysis, as well as by a comparison of its experimental ¹³C NMR chemical shifts with the corresponding population‐weighted values, as calculated by using DFT methods. Limitations of these used approaches became apparent but were overcome by combining the two methods.     

Measurement of 1H-1H residual dipolar coupling constants for structural studies of medium size molecules.

Residual dipolar coupling constants (RDCs) are being increasingly applied to elucidate the configuration and conformation of small organic molecules, peptides and oligosaccharides. In this paper we describe a set of robust 1D NMR methods for accurate and precise measurement of proton-proton RDCs of small and medium size molecules. The performance of these techniques is not impeded by the presence of overlapping and broad (1)H multiplets that are typically observed for such molecules in weakly aligned media. The use of these techniques provides access to a large pool of proton-proton RDCs opening new avenues for the solution structure elucidation of medium size molecules by NMR. The techniques are illustrated on the determination of the alignment tensor of the reducing monosaccharide ring of cellobiose and the determination of the relative configuration of sodium cholate.     

Simultaneous assignment of all diastereotopic protons in strychnine using RDCs: PELG as alignment medium for organic molecules

The concept of using residual dipolar couplings (RDCs) for the structure determination of organic molecules is applied to the simultaneous assignment of all diastereotopic protons in strychnine. To use this important NMR parameter the molecule has to be aligned in the magnetic field. Here we present a new alignment medium for organic substrates. The optimization of the alignment properties of mixtures of poly-gamma-ethyl-L-glutamate (PELG) and CDCl(3) are described and the alignment properties of PELG at different concentrations are evaluated. A comparison of PELG with poly-gamma-benzyl-L-glutamate (PBLG) shows considerable differences in the magnitude of alignment for strychnine in the two alignment media. PELG induces a lower degree of order and makes the measurement of residual dipolar couplings (RDCs) in strychnine possible. All one-bond C-H RDCs of strychnine in PELG were determined by using 2D heteronuclear single quantum coherence (HSQC) spectroscopy. The strategy for the extraction of RDCs for methylene groups is described in detail. The RDCs and order parameters are used to assign pairs of diastereotopic protons. This methodology can distinguish not only one pair of diastereotopic protons but it can be used to assign all pairs of diastereotopic protons simultaneously. Two different calculation approaches to achieve this task are described in detail.     

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 Residual Dipolar Couplings of Organic Molecules in Multiple Solvent Systems Using a Liquid‐Crystalline‐Based Medium

Residual dipolar coupling (RDC), a robust anisotropic NMR parameter for structural elucidation of organic molecules, is only accessible in an anisotropic environment. Herein, we introduce a novel alignment medium based on the molecular self‐assembly of oligopeptide amphiphile (OPA). This medium is compatible with different intermediate and polar solvent systems, such as CD₃OD, [D₆]DMSO, and D₂O. The preparation of the OPA‐based medium is simple and rapid, while only very weak background signals were observed from OPAs. Furthermore, we show that the purity of OPA has only a minor influence on the quality of the RDC data. These advantages allow RDC measurements of organic molecules with different polarities and solubilities with high efficiency and accuracy.     

Development of New Supramolecular Lyotropic Liquid Crystals and Their Application as Alignment Media for Organic Compounds

Most alignment media for the residual dipolar coupling (RDC) based molecular structure determination of small organic compounds consist of rod‐like polymers dissolved in organic solvents or of swollen cross‐linked polymer gels. Thus far, the synthesis of polymer‐based alignment media has been a challenging process, which is often followed by a time‐consuming sample preparation. We herein propose the use of non‐polymeric alignment media based on benzenetricarboxamides (BTAs), which self‐assemble into rod‐like supramolecules. Our newly found supramolecular lyotropic liquid crystals (LLCs) are studied in terms of their LLC properties and their suitability as alignment media in NMR spectroscopy. Scalable enantiodifferentiating properties are introduced through a sergeant‐and‐soldier principle by blending achiral with chiral substituted BTAs.     

Stretched poly(dimethylsiloxane) gels as NMR alignment media for apolar and weakly polar organic solvents: an ideal tool for measuring RDCs at low molecular concentrations

The measurement of residual dipolar couplings (RDCs), meanwhile a standard method for obtaining structural information in biomolecular NMR, requires partial alignment of the sample. Special demands on alignment media so far limit the applicability of this approach to small molecules in organic solvents. Major limitations are the free scalability of alignment and the suppression of residual signals of the alignment medium to allow effective measurement of low-concentration samples. Here, we present stretched poly(dimethylsiloxane) (PDMS) cross-linked by beta-rays as an alignment medium with no visible impurities in 1H NMR spectra but a single signal at approximately 0.1 ppm that can easily be removed by slightly modified water suppression methods. Besides the free scalability, its applicability to the measurement of RDCs in small molecules at low concentration is demonstrated on a approximately 12 mM sample of spiroindene. The induced alignment tensor in this case can be predicted reasonably well by a simplified model on the basis of steric interactions only.     

Assignment of phycocyanobilin in HMPT using triple resonance experiments

A complete assignment of all resonances of a small organic molecule is a prerequisite for a structure determination using NMR spectroscopy. This is conventionally obtained using a well‐established strategy based on COSY, HMQC and HMBC spectra. In case of phycocyanobilin (PCB) in HMPT this strategy was unsuccessful due to the symmetry of the molecule and extreme signal overlap. Since ¹³C and ¹⁵N labeled material was available, an alternative strategy for resonance assignment was used. Triple resonance experiments derived from experiments conventionally performed for proteins are sensitive and easy to analyze. Their application led to a complete and unambiguous assignment using three types of experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

A Self‐Assembled Oligopeptide as a Versatile NMR Alignment Medium for the Measurement of Residual Dipolar Couplings in Methanol

Residual dipolar coupling (RDC) is a powerful structural parameter for the determination of the constitution, conformation, and configuration of organic molecules. Herein, we report the first liquid crystal‐based orienting medium that is compatible with MeOH, thus enabling RDC acquisitions of a wide range of intermediate to polar organic molecules. The liquid crystals were produced from self‐assembled oligopeptide nanotubes (AAKLVFF), which are stable at very low concentrations. The presented alignment medium is highly homogeneous, and the size of RDCs can be scaled with the concentration of the peptide. To assess the accuracy of the RDC measurement by employing this new medium, seven bioactive natural products from different classes were chosen and analyzed. The straightforward preparation of the anisotropic alignment sample will offer a versatile and robust protocol for the routine RDC measurement of natural products.     

Direct structure determination of the self-condensation product of 1-phenylpentane-2,4-dione using T1 and NOE measurements from 13C NMR spectroscopy

The nuclear Overhauser effect (NOE) is known to depend on molecular dynamics and structure. However, in some cases the values obtained for selective homonuclear and heteronuclear NOE are much too small, considering that the nuclei involved are located within a short distance of each other in space. A quantitative treatment of the NOE values allows a clear explanation of this apparent anomaly, and allows the possibility of using T₁ and NOE values measured with broad-band proton irradiation. The corresponding relationship is useful for solving many structural problems in organic chemistry, and has the great advantage of employing the typical high resolution of fully decoupled spectra. The method was used in this work for the structure determination of the self-condensation product of 1-phenylpentane-2,4-dione, and it was concluded that the previous assignment of the ¹³C NMR spectrum was erroneous. An independent proof of the new assignment is given using the selective collapse of the fine structure under low-power irradiation of the methyl protons.     

Automatic perception of organic molecules based on essential structural information

Format conversion is very common in structure preparation in molecular modeling studies. Unfortunately, format conversion cannot always be executed precisely. We have developed an automatic method, called I-interpret (available on-line at http://www.sioc-ccbg.ac.cn/software/I-interpret/), for interpreting the chemical structure of a given organic molecule merely from its essential structural information, including element identities and three-dimensional coordinates of its component atoms. I-interpret uses standard geometrical parameters of organic molecules in atom/bond-type assignment. A series of elaborate considerations are arranged in a logical sequence for this purpose. I-interpret was tested on a set of 179 small organic molecules from the Protein Data Bank and a set of 1990 organic molecules from the NCI diversity set. On both sets, it achieved a success rate of over 95% in interpreting the correct chemical structures, outperforming other programs under our evaluation. I-interpret also provides users some optional functions, which makes it more flexible and powerful in practice. It may serve as a valuable tool for processing chemical structures in molecular modeling.     

A Two‐Tailed Phosphopeptide Crystallizes to Form a Lamellar Structure

The crystal structure of a designed phospholipid‐inspired amphiphilic phosphopeptide at 0.8 Å resolution is presented. The phosphorylated β‐hairpin peptide crystallizes to form a lamellar structure that is stabilized by intra‐ and intermolecular hydrogen bonding, including an extended β‐sheet structure, as well as aromatic interactions. This first reported crystal structure of a two‐tailed peptidic bilayer reveals similarities in thickness to a typical phospholipid bilayer. However, water molecules interact with the phosphopeptide in the hydrophilic region of the lattice. Additionally, solid‐state NMR was used to demonstrate correlation between the crystal structure and supramolecular nanostructures. The phosphopeptide was shown to self‐assemble into semi‐elliptical nanosheets, and solid‐state NMR provides insight into the self‐assembly mechanisms. This work brings a new dimension to the structural study of biomimetic amphiphilic peptides with determination of molecular organization at the atomic level.     

Measurement of Residual Dipolar Couplings of Organic Molecules in Multiple Solvent Systems Using a Liquid-Crystalline-Based Medium

Residual dipolar coupling (RDC), a robust anisotropic NMR parameter for structural elucidation of organic molecules, is only accessible in an anisotropic environment. Herein, we introduce a novel alignment medium based on the molecular self-assembly of oligopeptide amphiphile (OPA). This medium is compatible with different intermediate and polar solvent systems, such as CD₃OD, [D₆]DMSO, and D₂O. The preparation of the OPA-based medium is simple and rapid, while only very weak background signals were observed from OPAs. Furthermore, we show that the purity of OPA has only a minor influence on the quality of the RDC data. These advantages allow RDC measurements of organic molecules with different polarities and solubilities with high efficiency and accuracy.     

Orientational properties of stretched polystyrene gels in organic solvents and the suppression of their residual 1H NMR signals

While residual dipolar couplings (RDCs) are an established method in high-resolution biomolecular NMR, their use for structure determination of small molecules in organic solvents is limited by the alignment media available. Only recently stretched polystyrene (PS) gels were introduced for the measurement of RDCs on small compounds that allowed urgently needed free scalability of the induced anisotropy. Here, the properties of such stretched PS gels in different organic solvents as well as for different magnetic field strengths and temperatures are studied and practical NMR-spectroscopic aspects are discussed.     

Isotope‐Filtered 4D NMR Spectroscopy for Structure Determination of Humic Substances

Humic substances, the main component of soil organic matter, could form an integral part of green and sustainable solutions to the soil fertility problem. However, their global‐scale application is hindered from both scientific and regulatory perspectives by the lack of understanding of the molecular make‐up of these chromatographically inseparable mixtures containing thousands of molecules. Here we show how multidimensional NMR spectroscopy of isotopically tagged molecules enables structure characterization of humic compounds. We illustrate this approach by identifying major substitution patterns of phenolic aromatic moieties of a peat soil fulvic acid, an operational fraction of humic substances. Our methodology represents a paradigm shift in the use of NMR active tags in structure determination of small molecules in complex mixtures. Unlike previous tagging methodologies that focused on the signals of the tags, we utilize tags to directly probe the identity of the molecules they are attached to.     

Problems,artifacts and solutions in the INADEQUATE NMR experiment

The INADEQUATE experiment can provide unequalled, detailed information about the carbon skeleton of an organic molecule. However, it also has the reputation of requiring unreasonable amounts of sample. Modern spectrometers and probes have mitigated this problem, and it is now possible to get good structural data on a few milligrams of a typical organic small molecule. In this paper, we analyze the experiment step by step in some detail, to show how each part of the sequence can both contribute to maximum overall sensitivity and can lead to artifacts. We illustrate these methods on three molecules: 1‐octanol, the steroid 17α‐ethynylestradiol and the isoquinoline alkaloid β‐hydrastine. In particular, we show that not only is the standard experiment powerful, but also a version tuned to small couplings can contribute vital structural information on long‐range connectivities. If the delay in the spin echo is long, pairs of carbons with small couplings can create significant double‐quantum coherence and show correlations in the spectrum. These are two‐ and three‐bond correlations in a carbon chain or through a heteroatom in the molecule. All these mean that INADEQUATE can play a viable and important role in routine organic structure determination. Copyright © 2010 John Wiley & Sons, Ltd.     

Refinement of local and long-range structural order in theophylline-binding RNA using (13)C-(1)H residual dipolar couplings and restrained molecular dynamics.

13C-(1)H residual dipolar couplings (RDC) have been measured for the bases and sugars in the theophylline-binding RNA aptamer, dissolved in filamentous phage medium, and used to investigate the long-range structural and dynamic behavior of the molecule in the solution state. The orientation dependent RDC provide additional restraints to further refine the overall structure of the RNA-theophylline complex, whose long-range order was poorly defined in the NOE-based structural ensemble. Structure refinement using RDC normally assumes that molecular alignment can be characterized by a single tensor and that the molecule is essentially rigid. To address the validity of this assumption for the complex of interest, we have analyzed distinct domains of the RNA molecule separately, so that local structure and alignment tensors experienced by each region are independently determined. Alignment tensors for the stem regions of the molecule were allowed to float freely during a restrained molecular dynamics structure refinement protocol and found to converge to similar magnitudes. During the second stage of the calculation, a single alignment tensor was thus applied for the whole molecule and an average molecular conformation satisfying all experimental data was determined. Semirigid-body molecular dynamics calculations were used to reorient the refined helical regions to a relative orientation consistent with this alignment tensor, allowing determination of the global conformation of the molecule. Simultaneously, the local structure of the theophylline-binding core of the molecule was refined under the influence of this common tensor. The final ensemble has an average pairwise root mean square deviation of 1.50 +/- 0.19 A taken over all heavy atoms, compared to 3.5 +/- 1.1 A for the ensemble determined without residual dipolar coupling. This study illustrates the importance of considering both the local and long-range nature of RDC when applying these restraints to structure refinements of nucleic acids.     

Probing structure in invisible protein states with anisotropic NMR chemical shifts

A general method for obtaining quantitative structural information on invisible, excited protein states by solution-based NMR spectroscopy is presented. The approach exploits relaxation dispersion techniques in which changes in chemical shifts between ground and excited states are monitored in solutions with and without small amounts of residual molecular alignment. This allows the calculation of differences in chemical shifts induced by alignment that can be directly related to molecular structure, in cases where the orientation and magnitude of the chemical-shift tensor are well defined. An example using carbonyl chemical shifts as probes of a protein-ligand binding reaction is presented to illustrate and validate the method.