Determining the optimal size of small molecule mixtures for high throughput NMR screening

High-throughput screening (HTS) using NMR spectroscopy has become a common component of the drug discovery effort and is widely used throughout the pharmaceutical industry. NMR provides additional information about the nature of small molecule-protein interactions compared to traditional HTS methods. In order to achieve comparable efficiency, small molecules are often screened as mixtures in NMR-based assays. Nevertheless, an analysis of the efficiency of mixtures and a corresponding determination of the optimum mixture size (OMS) that minimizes the amount of material and instrumentation time required for an NMR screen has been lacking. A model for calculating OMS based on the application of the hypergeometric distribution function to determine the probability of a hit for various mixture sizes and hit rates is presented. An alternative method for the deconvolution of large screening mixtures is also discussed. These methods have been applied in a high-throughput NMR screening assay using a small, directed library.     

Systematic crosstalk in plasmalogen and diacyl lipid biosynthesis for their differential yet concerted molecular functions in the cell

Plasmalogen is a major phospholipid of mammalian cell membranes. Recently it is becoming evident that the sn-1 vinyl-ether linkage in plasmalogen, contrasting to the ester linkage in the counterpart diacyl glycerophospholipid, yields differential molecular characteristics for these lipids especially related to hydrocarbon-chain order, so as to concertedly regulate biological membrane processes. A role played by NMR in gaining information in this respect, ranging from molecular to tissue levels, draws particular attention. We note here that a broad range of enzymes in de novo synthesis pathway of plasmalogen commonly constitute that of diacyl glycerophospholipid. This fact forms the basis for systematic crosstalk that not only controls a quantitative balance between these lipids, but also senses a defect causing loss of lipid in either pathway for compensation by increase of the counterpart lipid. However, this inherent counterbalancing mechanism paradoxically amplifies imbalance in differential effects of these lipids in a diseased state on membrane processes. While sharing of enzymes has been recognized, it is now possible to overview the crosstalk with growing information for specific enzymes involved. The overview provides a fundamental clue to consider cell and tissue type-dependent schemes in regulating membrane processes by plasmalogen and diacyl glycerophospholipid in health and disease.     

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.     

Spatially encoded strategies in the execution of biomolecular-oriented 3D NMR experiments

Three-dimensional nuclear magnetic resonance (3D NMR) provides one of the foremost analytical tools available for the elucidation of biomolecular structure, function and dynamics. Executing a 3D NMR experiment generally involves scanning a series of time-domain signals S(t ₃), as a function of two time variables (t ₁, t ₂) which need to undergo parametric incrementations throughout independent experiments. Recent years have witnessed extensive efforts towards the acceleration of this kind of experiments. Among the different approaches that have been proposed counts an “ultrafast” scheme, which distinguishes itself from other propositions by enabling—at least in principle—the acquisition of the complete multidimensional NMR data set within a single transient. 2D protein NMR implementations of this single-scan method have been demonstrated, yet its potential for 3D acquisitions has only been exemplified on model organic compounds. This publication discusses a number of strategies that could make these spatial encoding protocols compatible with 3D biomolecular NMR applications. These include a merging of 2D ultrafast NMR principles with temporal 2D encoding schemes, which can yield 3D HNCO spectra from peptides and proteins within ≈100 s timescales. New processing issues that facilitate the collection of 3D NMR spectra by relying fully on spatial encoding principles are also assessed, and shown capable of delivering HNCO spectra within 1 s timescales. Limitations and prospects of these various schemes are briefly addressed.     

Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings

The feasibility of using two-bond ¹⁵N-¹H couplings to resolve the ¹H-¹H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example. The two-bond ¹⁵N-¹H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional ¹H-¹H-¹⁵N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension. The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure.     

The unusual internal motion of the villin headpiece subdomain

The thermostable 36‐residue subdomain of the villin headpiece (HP36) is the smallest known cooperatively folding protein. Although the folding and internal dynamics of HP36 and close variants have been extensively studied, there has not been a comprehensive investigation of side‐chain motion in this protein. Here, the fast motion of methyl‐bearing amino acid side chains is explored over a range of temperatures using site‐resolved solution nuclear magnetic resonance deuterium relaxation. The squared generalized order parameters of methyl groups extensively spatially segregate according to motional classes. This has not been observed before in any protein studied using this methodology. The class segregation is preserved from 275 to 305 K. Motions detected in Helix 3 suggest a fast timescale of conformational heterogeneity that has not been previously observed but is consistent with a range of folding and dynamics studies. Finally, a comparison between the order parameters in solution with previous results based on solid‐state nuclear magnetic resonance deuterium line shape analysis of HP36 in partially hydrated powders shows a clear disagreement for half of the sites. This result has significant implications for the interpretation of data derived from a variety of approaches that rely on partially hydrated protein samples.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

The combined use of selective deuteration and double resonance experiments in assigning the 1H resonances of valine and tyrosine residues of dihydrofolate reductase

Selective deuteration is a general solution to the resolution problem which limits the application of double resonance experiments to the assignment of the ¹H NMR spectra of proteins. Spin-decoupling and NOE experiments have been carried out on Lactobacillus casei dihydrofolate reductase and on selectively deuterated derivatives of the enzyme containing either [γ-²H₆]Val or (α,δ₂,?₁-²H₃]His, [α,δ₁,δ₂,?₁,?₂,ζ-²H₆]Phe, [α,δ₁,?₃,ζ₂,ζ₃,η₂-²H₆]Trp and [α,?₁,?₂-²H₃]Tyr. When combined with ring-current shift calculations based on the crystal structure of the enzyme, these experiments allow us to assign ¹H resonances of Val 61, Val 115, Tyr 46 and Tyr 68.     

Tools for the automated assignment of high-resolution three-dimensional protein NMR spectra based on pattern recognition techniques

One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assignment tool based onpattern recognition techniques. This tool employs user-specified templatesto search for patterns of peaks in the original spectra; these patterns maycorrespond to side-chain or backbone fragments. Multiple spectra willnormally be searched simultaneously to reduce the impact of noise. Thesearch generates a preliminary list of putative assignments, which arefiltered by a set of heuristic algorithms to produce the final results list.Each result contains a set of chemical shift values plus information aboutthe peaks found. The results may be used as input for combinatorialroutines, such as sequential assignment procedures, in place of peak lists.Two examples are presented, in which (i) HCCH-COSY and -TOCSY spectra arescanned for side-chain spin systems; and (ii) backbone spin systems aredetected in a set of spectra comprising HNCA, HN(CO)CA, HNCO, HN(CA)CO,CBCANH and CBCA(CO)NH.     

PR-CALC: A Program for the Reconstruction of NMR Spectra from Projections

Projection-reconstruction NMR (PR-NMR) has attracted growing attention as a method for collecting multidimensional NMR data rapidly. The PR-NMR procedure involves measuring lower-dimensional projections of a higher-dimensional spectrum, which are then used for the mathematical reconstruction of the full spectrum. We describe here the program PR-CALC, for the reconstruction of NMR spectra from projection data. This program implements a number of reconstruction algorithms, highly optimized to achieve maximal performance, and manages the reconstruction process automatically, producing either full spectra or subsets, such as regions or slices, as requested. The ability to obtain subsets allows large spectra to be analyzed by reconstructing and examining only those subsets containing peaks, offering considerable savings in processing time and storage space. PR-CALC is straightforward to use, and integrates directly into the conventional pipeline for data processing and analysis. It was written in standard C+ + and should run on any platform. The organization is flexible, and permits easy extension of capabilities, as well as reuse in new software. PR-CALC should facilitate the widespread utilization of PR-NMR in biomedical research. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

黄牛木属植物的化学成分与生物活性研究进展

本文综述了从1963年以来黄牛木属植物的化学成分和生物活性研究,概括了该属植物中部分口山酮和蒽醌化合物的NMR数据。     

Synthesis and nuclear magnetic resonance study of 3-dehydroecdysteroids

Several 3-dehydro- (or 3-oxo-) ecdysteroids have been prepared by enzymatic and/or chemical means. Methods for their purification using various high-performance liquid chromatography systems are described. Proton and carbon nuclear magnetic resonance analyses show that 3-dehydroecdysteroids when dissolved in water or methanol (but not in chloroform) present a temperature-dependent equilibrium between two forms. The possible structure of these two forms is discussed.     

珙桐叶中喹啉类生物碱甙——pumiloside结构鉴定

首次从珙桐叶甲醇提取物的水溶性部分得到一配糖体化合物-喹啉类生物碱甙(pumiloside),它是抗肿瘤物质喜树碱的生物合成前体。本文对其结构进行鉴定。     

Eremophilane-type glucosides from the leaves of Ligularia calthifolia Maxim

Three eremophilane-type glucosides (1–3) along with three known congeners alticolosides A, B and D (4–6) have been isolated from the leaves of Ligularia calthifolia Maxim. (Asteraceae). The structures of 1–3 have been elucidated by spectroscopic analysis as well as by chemical transformations. A distinctive feature of the compounds 1–3 is the presence of an acetyl group in the glucose residues. The compounds were non-cytotoxic against human normal prostate cells as well as prostate cancer cells.     

Nucleotide-amino acid interactions and their relation to the genetic code

Summary The apparent dissociation constants of the complexes of AMP with the methyl esters of amino acids in aqueous solution exhibit good correlations with features of the genetic code and with the frequencies of occurrence of amino acid residues in proteins. Thus it is likely that chemically selective nucleotide-amino acid interactions were involved in the processes of chemical evolution that have led to the emergence of the genetic code. Based on these correlations a storage device for the information regarding nucleotide-amino acid interactions is proposed. It involves processes of simultaneous polymerization to polynucleotides and polypeptides.     

Molecular mechanism of the interaction between MDM2 and p53

We have investigated the kinetic and thermodynamic basis of the p53-MDM2 interaction using a set of peptides based on residues 15-29 of p53. Wild-type p53 peptide bound MDM2 with a dissociation constant of 580nM. Phosphorylation of S15 and S20 did not affect binding, but T18 phosphorylation weakened binding tenfold, indicating that phosphorylation of only T18 is responsible for abrogating p53-MDM2 binding. Truncation to residues 17-26 increased affinity 13-fold, but further truncation to 19-26 abolished binding. NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure of MDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.