NUScon: a community-driven platform for quantitative evaluation of nonuniform sampling in NMR.

Although the concepts of nonuniform sampling (NUS​​​​​​​) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago , it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection of closely spaced peaks (i.e., "resolution") or peaks of weak intensity (i.e., "sensitivity"). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the "Nonuniform Sampling Contest" (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform.

Nanomedical Relevance of the Intermolecular Interaction Dynamics-Examples from Lysozymes and Insulins.

Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.

Complete protein assignment from sets of spectra recorded overnight.

A flexible and scalable approach for protein NMR is introduced that builds on rapid data collection via projection spectroscopy and analysis of the spectral input data via joint decomposition. Input data may originate from various types of spectra, depending on the ultimate goal: these may result from experiments based on triple-resonance pulse sequences, or on TOCSY or NOESY sequences, or mixtures thereof. Flexible refers to the free choice of spectra for the joint decompositions depending on the purpose: assignments, structure, dynamics, interactions. Scalable means that the approach is open to the addition of similar or different experiments, e.g. larger proteins may require a wider selection of triple-resonance based experiments. Central to the proposed approach is the mutual support among the different spectra during the spectral analysis: for example, sparser triple-resonance spectra may help decomposing (separating) spin systems in a TOCSY or identifying unique NOEs. In the example presented, backbone plus side chain assignments of ubiquitin were obtained from the combination of either two or three of the following projection experiments: a 4D HCCCONH, a 4D HNCACO and a 3D HNCACB. In all cases, TOCSY data (4D HCCCONH) proved crucial not only for the side chain assignments, but also for the sequential assignment. Even when total recording time was reduced to about 10 h, nearly complete assignments were obtained, with very few missing assignments and even fewer differences to a reference.

Lysozyme's lectin-like characteristics facilitates its immune defense function.

Interactions between human lysozyme (HL) and the lipopolysaccharide (LPS) of Klebsiella pneumoniae O1, a causative agent of lung infection, were identified by surface plasmon resonance. To characterize the molecular mechanism of this interaction, HL binding to synthetic disaccharides and tetrasaccharides representing one and two repeating units, respectively, of the O-chain of this LPS were studied. pH-dependent structural rearrangements of HL after interaction with the disaccharide were observed through nuclear magnetic resonance. The crystal structure of the HL-tetrasaccharide complex revealed carbohydrate chain packing into the A, B, C, and D binding sites of HL, which primarily occurred through residue-specific, direct or water-mediated hydrogen bonds and hydrophobic contacts. Overall, these results support a crucial role of the Glu35/Asp53/Trp63/Asp102 residues in HL binding to the tetrasaccharide. These observations suggest an unknown glycan-guided mechanism that underlies recognition of the bacterial cell wall by lysozyme and may complement the HL immune defense function.

DIADECOMP: A new approach to analyze decompositions from projection spectroscopy.

We demonstrate for the first time a complete small protein characterization with the projection-decomposition approach, including full assignments as well as determination of the 3D fold. In TOCSY- and NOESY-type 4D experiments, pairing of signals from hydrogens and from their respective heavy atoms in decompositions represents a new problem. An approach, referred to as "DIADECOMP" (diagonal decomposition), is introduced to solve this problem; it consists of two separate decompositions of the input projections, differing in a 45° rotation of the spectral axes. While DIADECOMP requires a somewhat complex formulation, in practice it results in observing signals in the rotated decompositions that correspond to sums or differences of frequencies. When applied to a small protein, human defensin ß6, the analysis of a HCC(CO)NH-TOCSY with DIADECOMP results in largely unambiguous assignments of the aliphatic side chain groups. Furthermore, DIADECOMP applied to a 15N-HSQC-NOESY-15N-HSQC provides all expected short distances between amide groups (defined as all HN-HN distances <3.5Å in a reference structure). It is worth noting that short HN-HN distances unambiguously define α-helices, the alignment of ß-strands in sheets, as well as the presence of ß-bulges. This approach of using a minimal amount of NMR data, namely four projection experiments recorded in ∼2.5days, resulted for the human defensin ß6 in complete assignments and a backbone fold with a RMSD of the non-flexible structure of 0.6Å. Uniqueness of decompositions specifically from TOCSY- and NOESY-type 4D experiments is discussed.

Molecular Basis of the Receptor Interactions of Polysialic Acid (polySia), polySia Mimetics, and Sulfated Polysaccharides.

Polysialic acid (polySia) and polySia glycomimetic molecules support nerve cell regeneration, differentiation, and neuronal plasticity. With a combination of biophysical and biochemical methods, as well as data mining and molecular modeling techniques, it is possible to correlate specific ligand-receptor interactions with biochemical processes and in vivo studies that focus on the potential therapeutic impact of polySia, polySia glycomimetics, and sulfated polysaccharides in neuronal diseases. With this strategy, the receptor interactions of polySia and polySia mimetics can be understood on a submolecular level. As the HNK-1 glycan also enhances neuronal functions, we tested whether similar sulfated oligo- and polysaccharides from seaweed could be suitable, in addition to polySia, for finding potential new routes into patient care focusing on an improved cure for various neuronal diseases. The knowledge obtained here on the structural interplay between polySia or sulfated polysaccharides and their receptors can be exploited to develop new drugs and application routes for the treatment of neurological diseases and dysfunctions.

A consensus on protein structure accuracy in NMR?

The precision of an NMR structure may be manipulated by calculation parameters such as calibration factors. Its accuracy is, however, a different issue. In this issue of Structure, Buchner and Güntert present "consensus structure bundles," where precision analysis allows estimation of accuracy.

De novo tertiary structure prediction using RNA123--benchmarking and application to Macugen.

The present benchmarking study utilizes the RNA123 program for de novo prediction of tertiary structures of a set of 50 RNA molecules for which X-ray/NMR structures are available, based on the nucleic acid sequence only. All molecules contain a hairpin loop motif and a helical structure of canonical and non-canonical base pairs, interrupted by bulges and internal loops to various degrees. RNA molecules with double helices made up purely by canonical base pairing, and molecules containing symmetric internal loops of non-canonical base pairing are, overall, very well predicted. Structures containing bulges and asymmetric internal loops, and more complex structures containing multiple bulges and internal loops in the same molecule, result in larger deviations from their X-ray/NMR predicted structures due to higher degree of flexibility of the nucleotide bases in these regions. In a majority of the molecules included herein, the RNA123 program was, however, able to predict the tertiary structure with a heavy atom RMSD of less than 5 Å to the X-ray/NMR structure, and the models were in most cases structurally closer to the X-ray/NMR structures than models predicted by MC-Fold and MC-Sym. A set of RNA molecules containing pseudoknot tertiary structure motifs were included, but neither of the programs was able to predict the folding of the single-stranded stem onto the helix without additional structural input. The RNA123 program was then applied to predict the tertiary structure of the RNA segment of Macugen®, the first RNA aptamer approved for clinical use, and for which no tertiary structure has yet been solved. Four possible tertiary structures were predicted for this 27-nucleic-acid-long RNA molecule, which will be used in constructing a full model of the PEGylated aptamer and its interaction with the vascular endothelial growth factor target.

Minor-groove binding drugs: where is the second Hoechst 33258 molecule?

Hoechst 33258 binds with high affinity into the minor groove of AT-rich sequences of double-helical DNA. Despite extensive studies of this and analogous DNA binding molecules, there still remains uncertainty concerning the interactions when multiple ligand molecules are accommodated within close distance. Albeit not of direct concern for most biomedical applications, which are at low drug concentrations, interaction studies for higher drug binding are important as they can give fundamental insight into binding mechanisms and specificity, including drug self-stacking interactions that can provide base-sequence specificity. Using circular dichroism (CD), isothermal titration calorimetry (ITC), and proton nuclear magnetic resonance ((1)H NMR), we examine the binding of Hoechst 33258 to three oligonucleotide duplexes containing AT regions of different lengths: [d(CGCGAATTCGCG)]2 (A2T2), [d(CGCAAATTTGCG)]2 (A3T3), and [d(CGAAAATTTTCG)]2 (A4T4). We find similar binding geometries in the minor groove for all oligonucleotides when the ligand-to-duplex ratio is less than 1:1. At higher ratios, a second ligand can be accommodated in the minor groove of A4T4 but not A2T2 or A3T3. We conclude that the binding of the second Hoechst to A4T4 is not cooperative and that the molecules are sitting with a small separation apart, one after the other, and not in a sandwich structure as previously proposed.

Initial DNA interactions of the binuclear threading intercalator Λ,Λ-[µ-bidppz(bipy)4Ru2]4+: an NMR study with [d(CGCGAATTCGCG)]2.

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one Ru(II), through the DNA base-pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear Ru(II) compounds and DNA. We report a structural NMR study on the binuclear Ru(II) intercalator Λ,Λ-B (Λ,Λ-[µ-bidppz(bipy)4Ru2](4+); bidppz = 11,11'-bis(dipyrido[3,2-a:2',3'-c]phenazinyl, bipy = 2,2'-bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]2. Threading of Λ,Λ-B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ-B is trapped in a well-defined surface-bound state consisting of an eccentric minor-groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ-B. Molecular dynamics simulations show that this minor-groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru(2+) moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ-B with natural calf thymus DNA. The present minor-groove binding mode is proposed to represent the initial surface interactions of binuclear Ru(II) compounds prior to intercalation into AT-rich DNA.

Sequence and immunogenicity of a clinically approved novel measles virus vaccine vector.

The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 10 ( 20) . The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors.

Automated protein backbone assignment using the projection-decomposition approach.

Spectral projection experiments by NMR in conjunction with decomposition analysis have been previously introduced for the backbone assignment of proteins; various pulse sequences as well as the behaviour with low signal-to-noise or chemical shift degeneracy have been illustrated. As a guide for routine applications of this combined tool, we provide here a systematic analysis on different types of proteins using welldefined run-time parameters. As a second result of this study, the backbone assignment module SHABBA was extensively rewritten and improved. Calculations on ubiquitin yielded again fully correct and nearly complete backbone and CHß assignments. For the 128 residue long azurin, missing assignments mostly affect Hα and Hß. Among the remaining backbone (plus Cß) nuclei 97.5 % could be assigned with 1.0 % differences to a reference. Finally, the new SHABBA algorithm was applied to projections recorded for a yeast histone protein domain at room temperature, where the protein is subject to partial unfolding: this leads to unobservable resonances (about a dozen missing signals in a normal 15N-HSQC) and extensive degeneracy among the resonances. From the clearly observable residues, 97.5 % of the backbone and CHßresonances could be assigned, of which only 0.8 % showed differences to published shifts. An additional study on the protein MMP20, which exhibits spectral difficulties to an even larger extent, explores the limitations of the approach.

TSAR: a program for automatic resonance assignment using 2D cross-sections of high dimensionality, high-resolution spectra.

While NMR studies of proteins typically aim at structure, dynamics or interactions, resonance assignments represent in almost all cases the initial step of the analysis. With increasing complexity of the NMR spectra, for example due to decreasing extent of ordered structure, this task often becomes both difficult and time-consuming, and the recording of high-dimensional data with high-resolution may be essential. Random sampling of the evolution time space, combined with sparse multidimensional Fourier transform (SMFT), allows for efficient recording of very high dimensional spectra (≥4 dimensions) while maintaining high resolution. However, the nature of this data demands for automation of the assignment process. Here we present the program TSAR (Tool for SMFT-based Assignment of Resonances), which exploits all advantages of SMFT input. Moreover, its flexibility allows to process data from any type of experiments that provide sequential connectivities. The algorithm was tested on several protein samples, including a disordered 81-residue fragment of the δ subunit of RNA polymerase from Bacillus subtilis containing various repetitive sequences. For our test examples, TSAR achieves a high percentage of assigned residues without any erroneous assignments.

Solution structure and biophysical properties of MqsA, a Zn-containing antitoxin from Escherichia coli.

The gene ygiT (mqsA) of Escherichia coli encodes MqsA, the antitoxin of the motility quorum sensing regulator (MqsR). Both proteins are considered to form a DNA binding complex and to be involved in the formation of biofilms and persisters. We have determined the three-dimensional solution structure of MqsA by high-resolution NMR. The protein comprises a well-defined N-terminal domain with a Zn finger motif usually found in eukaryotes, and a defined C-terminal domain with a typical prokaryotic DNA binding helix-turn-helix motif. The two well-defined domains of MqsA have almost identical structure in solution and in the two published crystal structures of dimeric MqsA bound to either MqsR or DNA. However, the connection of the two domains with a flexible linker yields a large variety of possible conformations in solution, which is not reflected in the crystal structures. MqsA binds Zn with all four cysteines, a stoichiometry of 1:1 and a femtomolar affinity (K(a)≥ 10¹7M⁻¹ at 23°C, pH 7.0).

Structural characterisation of a histone domain by projection-decomposition.

We demonstrate that two projection experiments, a (15)N-HSQC-NOESY-(15)N-HSQC and a (13)C-HSQC-NOESY-(15)N-HSQC, recorded for a histone domain from yeast, contain enough information to support a structural characterisation of the protein. At the temperature used, 298 K, the histone domain exhibits a very high extent of chemical shift degeneracy that is uncharacteristic for a fully folded domain. Nonetheless, a structured core of 67 residues, which is formed by three α-helices and a two-stranded ß-sheet is defined by this NOESY data; this core structure was shown earlier to be present at lower temperature. The above two experiments, which together required 18 h of instrument time, are part of a set of five projection experiments acquired during 2.5 days with the goal of complete characterisation of proteins, including full resonance assignment and structure.