Sequential Assignment Method Research Articles

Covariate Balancing Methods for Randomized Controlled Trials Are Not Adversarially Robust.

The first step toward investigating the effectiveness of a treatment via a randomized trial is to split the population into control and treatment groups then compare the average response of the treatment group receiving the treatment to the control group receiving the placebo. To ensure that the difference between the two groups is caused only by the treatment, it is crucial that the control and the treatment groups have similar statistics. Indeed, the validity and reliability of a trial are determined by the similarity of two groups' statistics. Covariate balancing methods increase the similarity between the distributions of the two groups' covariates. However, often in practice, there are not enough samples to accurately estimate the groups' covariate distributions. In this article, we empirically show that covariate balancing with the standardized means difference (SMD) covariate balancing measure, as well as Pocock and Simon's sequential treatment assignment method, are susceptible to worst case treatment assignments. Worst case treatment assignments are those admitted by the covariate balance measure, but result in highest possible ATE estimation errors. We developed an adversarial attack to find adversarial treatment assignment for any given trial. Then, we provide an index to measure how close the given trial is to the worst case. To this end, we provide an optimization-based algorithm, namely adversarial treatment assignment in treatment effect trials (ATASTREET), to find the adversarial treatment assignments.

Online Sequential Task Assignment With Execution Uncertainties for Multiple Robot Manipulators

In order to let multiple robot manipulators cooperatively complete a sequence of tasks in a shared workspace under task execution uncertainty, this letter proposes a multi-robot task allocation framework for constantly assigning tasks to robots, while the interference among concurrent robot motions is account for. An online sequential task assignment method is presented, which decouples the time-extended problem into a sequence of synchronous and asynchronous instantaneous assignment sub-problems. This renders the approach capable of reacting to task execution uncertainties in real-time. A one-step-ahead simulation method is employed to reduce the idle time of robots and improve task completion efficiency. Each instantaneous assignment sub-problem is modeled as an optimal assignment problem with variable utility and solved by a branch-and-bound algorithm, with which multi-robot motion coordination is integrated. Experimental results conducted with three Franka-Emika Panda arms show that these can cooperatively complete all tasks without collision and little waiting time. Simulations with larger multi-robot systems show that the approach scales linearly with the number of robots.

Crowdsourcing Software Task Assignment Method for Collaborative Development

Software crowdsourcing is an emerging and promising software development model. It is based on the characteristics of Internet community intelligence, which makes it have certain advantages in development cost and product quality. Companies are increasingly using crowdsourcing to accomplish specific software development tasks. However, this development model still faces many challenges. One of the key issues is the collaboration between crowdsourced workers. Developer collaboration is important to software development, but workers in crowdsourcing come from an undefined network community, so it's hard to guarantee that they can work together. This paper focuses on task assignment and uses the active time of workers as the basis of grouping to provide a solution for multi-task to multi-worker allocation. Based on the on-demand distribution model, this paper considers three factors: worker's ability, task module complexity, and worker's active time. First, the workers are divided into multiple collaborative candidate groups based on active time. Then, the Hungarian algorithm is used to select the optimal workers for each module from the collaborative candidate groups of each task, and the coordination candidate group replacement strategy is used to solve the assignment failure problem. Finally completing the assignment of all tasks within an assignment cycle. The experiments have shown that the proposed method increases the total utility by 25% and the success rate of distribution by 30% than the sequential assignment method. The proposed method can give a reasonable solution for software crowdsourcing task allocation based on collaborative development.

Advances in stable isotope assisted labeling strategies with information science

Stable-isotope (SI) labeling of proteins is an essential technique to investigate their structures, interactions or dynamics by nuclear magnetic resonance (NMR) spectroscopy. The assignment of the main-chain signals, which is the fundamental first step in these analyses, is usually achieved by a sequential assignment method based on triple resonance experiments. Independently of the triple resonance experiment-based sequential assignment, amino acid-selective SI labeling is beneficial for discriminating the amino acid type of each signal; therefore, it is especially useful for the signal assignment of difficult targets. Various combinatorial selective labeling schemes have been developed as more sophisticated labeling strategies. In these strategies, amino acids are represented by combinations of SI labeled samples, rather than simply assigning one amino acid to one SI labeled sample as in the case of conventional amino acid-selective labeling. These strategies have proven to be useful for NMR analyses of difficult proteins, such as those in large complex systems, in living cells, attached or integrated into membranes, or with poor solubility. In this review, recent advances in stable isotope assisted labeling strategies will be discussed.

NMR spectral analysis using prior knowledge

Signal assignment is a fundamental step for analyses of protein structure and dynamics with nuclear magnetic resonance (NMR). Main-chain signal assignment is achieved with a sequential assignment method and/or an amino-acid selective stable isotope labeling (AASIL) method. Combinatorial selective labeling (CSL) methods, as well as our labeling strategy, stable isotope encoding (SiCode), were developed to reduce the required number of labeled samples, since one of the drawbacks of AASIL is that many samples are needed. Signal overlapping in NMR spectra interferes with amino-acid determination by CSL and SiCode. Since spectral deconvolution by peak fitting with a gradient method cannot resolve closely overlapped signals, we developed a new method to perform both peak fitting and amino acid determination simultaneously, with a replica exchange Monte Carlo method, incorporating prior knowledge of stable-isotope labeling ratios and the amino-acid sequence of the protein.

Multimodal Evacuation Simulation and Scenario Analysis in Dense Urban Area

Simulation is an effective tool for estimating evacuation times, identifying system bottlenecks, and evaluating traffic management strategies under a variety of operational and behavioral assumptions. The development of a multimodal simulation model for modeling evacuation activities in Center City, Philadelphia, Pennsylvania, is presented. The model applied a dynamic sequential assignment method to simulate the movement and interactions of pedestrians, private vehicles, and buses while considering the variation of evacuation demand and network conditions during evacuation operations. The model was used to perform scenario analysis and provide inputs to update current evacuation plans. This study shares the experience and findings from the Philadelphia study in modeling multimodal evacuations in dense urban areas.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Enzymatic Synthesis of New Oligosaccharides Using Mannosyltransferases from Candida Species and Their NMR Assignments

The outer layer of the cell wall of pathogenic fungi, Candida species, consists of mannan, which plays an important role in infection. In this study, we synthesized several oligosaccharides using mannosyltransferases obtained from Candida parapsilosis and Candida albicans. Namely, we synthesized mannotetraoses [Manalpha1--> 2Manalpha1-->3Manalpha1-->2Man and Manalpha1-->3(Manalpha1-->6)Manalpha1-->2Man] from mannotriose, Manalpha1-->3Manalpha1--> 2Man, and mannohexaoses [Manalpha1-->2Manalpha1-->3Manalpha1-->3Manalpha1-->2Manalpha1-->2Man and Manalpha1-->3(Manalpha1-->6)Manalpha1-->3Manalpha1-->2Manalpha1-->2Man] from mannopentaose, Manalpha1-->3Manalpha1-->3Manalpha1-->2Manalpha1-->2Man. The linkage sequence of these oligosaccharides was identified by a sequential (1)H-NMR assignment method combined with rotating frame nuclear Overhauser enhancement spectroscopy and relayed coherence transfer spectroscopy. The steric effect by the alpha-1,6-linked branching mannose residue to the H-1 proton chemical shift of the neighboring 3-O-substituted mannose residue was different from that of the 2-O-substituted mannose residue. These oligosaccharides having novel structures seem to be useful as the substrate or ligand for glycomics.

Asynchronous Forward-checking for DisCSPs

A new search algorithm for solving distributed constraint satisfaction problems (DisCSPs) is presented. Agents assign variables sequentially, but perform forward checking asynchronously. The asynchronous forward-checking algorithm (AFC) is a distributed search algorithm that keeps one consistent partial assignment at all times. Forward checking is performed by sending copies of the partial assignment to all unassigned agents concurrently. The algorithm is described in detail and its correctness proven. The sequential assignment method of AFC leads naturally to dynamic ordering of agents during search. Several ordering heuristics are presented. The three best heuristics are evaluated and shown to improve the performance of AFC with static order by a large factor. An experimental comparison of AFC to asynchronous backtracking (ABT) on randomly generated DisCSPs is also presented. AFC with ordering heuristics outperforms ABT by a large factor on the harder instances of random DisCSPs. These results hold for two measures of performance: number of non-concurrent constraints checks and number of messages sent.

Site-directed Mutagenesis and Kinetic Studies of the West Nile Virus NS3 Protease Identify Key Enzyme-Substrate Interactions

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.

NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands

Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe a strategy for resonance assignment in selectively labeled proteins. Our approach involves a spin-labeled analog of a ligand of which the three-dimensional structure in complex with the target protein is known. Other methods for introduction of the spin label are possible. The paramagnetic center causes faster relaxation of all neighboring nuclei in a distance-dependent manner. Measurement of this effect allows to deduce distances between isotopically labeled residues and the paramagnetic center which can be used for resonance assignment. The method is demonstrated for the catalytic domain of Abl kinase in complex with the inhibitor, STI571.

On the span in channel assignment problems: bounds, computing and counting

The channel assignment problem involves assigning radio channels to transmitters, using a small span of channels but without causing excessive interference. We consider a standard model for channel assignment, the constraint matrix model, which extends ideas of graph colouring. Given a graph G=( V, E) and a length l( uv) for each edge uv of G, we call an assignment φ: V→{1,…, t} feasible if | φ( u)− φ( v)|⩾ l( uv) for each edge uv. The least t for which there is a feasible assignment is the span of the problem. We first derive two bounds on the span, an upper bound (from a sequential assignment method) and a lower bound. We then see that an extension of the Gallai-Roy theorem on chromatic number and orientations shows that the span can be calculated in O( n!) steps for a graph with n nodes, neglecting a polynomial factor. We prove that, if the edge-lengths are bounded, then we may calculate the span in exponential time, that is, in time O( c n ) for a constant c. Finally we consider counting feasible assignments and related quantities.

NMR spectroscopy: a multifaceted approach to macromolecular structure.

1. Introduction 292. Landmarks in NMR of macromolecules 322.1 Protein structures and methods development 322.1.1 Sequential assignment method 322.1.2 Triple-resonance experiments 342.1.3 Structures of large proteins 362.2 Protein–nucleic acid complexes 372.3 RNA structures 382.4 Membrane-bound systems 393. NMR spectroscopy today 403.1 State-of-the-art structure determination 413.2 New methods 443.2.1 Residual dipolar couplings 443.2.2 Direct detection of hydrogen bonds 443.2.3 Spin labeling 453.2.4 Segmental labeling 463.3 Protein complexes 473.4 Mobility studies 503.5 Determination of time-dependent structures 523.6 Drug discovery 534. The future of NMR 544.1 The ease of structure determination 544.2 The ease of making recombinant protein 554.3 Post-translationally modified proteins 554.4 Approaches to large and/or membrane-bound proteins 564.5 NMR in structural genomics 564.6 Synergy of NMR and crystallography in protein structure determination 565. Conclusion 576. Acknowledgements 577. References 57Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dötsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

Sequential eigenstructure assignment with minimal state‐feedback gain†

This paper proposes a practical and sequential eigenstructure assignment method for designing a parametric state‐feedback controller in multi‐input linear time‐invariant systems. This approach, which sequentially shifts a prescribed set of self ‐conjugate multiple eigenvalues to the closed‐loop system, can explicitly parameterize in groups, and is very useful. Spectrum‐invariant free parameters are proposed to simultaneously deal with two problems: (i) assigning the desired corresponding eigenvectors and generalized eigenvectors within an admissible class, and (ii) minimizing the Frobenius norm of the resulting controller gain. The shifting of the uncontrollable mode is discussed in some detail. A scheme for determining the minimum Frobenious norm of the resultant state‐feedback matrix gain is developed. Finally, a numerical example is used for illustration of the proposed method.

NMR study of the galactomannans of Trichophyton mentagrophytes and Trichophyton rubrum.

Around 90% of chronic dermatophyte infections are caused by the fungi Trichophyton mentagrophytes and Trichophyton rubrum. One of the causes of the chronic infection resides in the immunosuppressive effects of the cell-wall components of these organisms. Therefore we have attempted to identify the chemical structure of galactomannan, one of the major cell-wall components. The cell-wall polysaccharides secreted by T. mentagrophytes and T. rubrum were isolated from the culture medium and fractionated into three subfractions by DEAE-Sephadex chromatography. Analysis of each subfraction by NMR indicated that there are two kinds of polysaccharides present, i.e. mannan and galactomannan. The mannan has a linear backbone consisting of alpha1,6-linked mannose units, with alpha1,2-linked mannose units as side chains. The core mannan moiety of the galactomannan was analysed by a sequential NMR assignment method after removing the galactofuranose units by acid treatment. The result indicates that the mannan moiety has a linear repeating structure of alpha1,2-linked mannotetraose units connected by an alpha1,6 linkage. The H-1 signals of the two intermediary alpha1, 2-linked mannoses of the tetraose unit showed a significant upfield shift (Deltadelta=0.05-0.08 p.p.m.), due to the steric effect of an alpha1,6-linked mannose unit. The attachment point of the galactofuranose units was determined at C-3 of the core mannan by the assignment of the downfield-shifted 13C signals of the galactomannan compared with those of the acid-modified product. In these galactomannans there were no polygalactofuranosyl chains which have been found in Penicillium charlesii and Aspergillus fumigatus.

The solution structure of desulforedoxin, a simple iron-sulfur protein

Desulforedoxin is a simple dimeric protein isolated from Desulfovibriogigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with a 36-amino-acid monomer). 1H NMR spectra of the oxidized Dx(Fe3+) and reduced Dx(Fe2+) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to Hβ protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel β-sheet running from D5 to V18 with a well-defined β-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dx monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NHe amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22–T25, was observed. Comparison between the Fe and Zn forms of Dx suggests that metal substitution does not have an effect on the structure of the protein.

NMR solution structure of the C-terminal fragment 255-316 of thermolysin: a dimer formed by subunits having the native structure.

The solution structure of the C-terminal fragment 255-316 of thermolysin has been determined by two-dimensional proton NMR methods. For this disulfide-free fragment there was a previous proposal according to which it would fold into a stable helical structure forming a dimer at concentrations above 0.06 mM. A complete assignment of the proton NMR resonances of the backbone and amino acid side chains of the fragment was first performed using standard sequential assignment methods. On the basis of 729 distance constraints derived from unambiguously assigned nuclear Overhauser effect (NOE) proton connectivities, the three-dimensional structure of a monomeric unit was then determined by using distance geometry and restrained molecular dynamic methods. The globular structure of fragment 255-316 of thermolysin in solution, composed of three helices, is largely coincident with that of the corresponding region in the crystallographic structure of intact thermolysin [Holmes, M. A., & Matthews, B. W. (1982) J. Mol. Biol. 160, 623-639]. This fact allowed identification as intersubunit of up to 52 NOE cross correlations, which were used to dock the two subunits into a symmetric dimer structure. The obtained dimeric structure served as the starting structure in a final restrained molecular dynamic calculation subjected to a total of 1562 distance constraints. In the resulting dimeric structure, the interface between the two subunits, of a marked hydrophobic character, coincides topologically with the one between the 255-316 fragment and the rest of the protein in the intact enzyme. The present work decisively shows that the thermolysin fragment 255-316 can attain a stable and nativelike structure independently of the rest of the polypeptide chain. Considering that the thermolysin molecule is constituted of two structural domains of equal size (residues 1-157 and 158-316), the results of this study show that autonomously folding units can be substantially smaller than entire domains.

Molecular Design of DNA-Directed Ligands with Specific Interactions: Solution NMR Studies of the Interaction of a m-Hydroxy Analog of Hoechst 33258 with d(CGCGAATTCGCG)2

We have used one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance spectroscopy at 600 MHz for structural analysis of the complex formed between d(CGCGAATTCGCG)2 and 2-[2-(3-hydroxyphenyl)-6-benzimidazoyl]-6-(1-methyl-4-piperazinyl) benzimidazole (meta-Hoechst). This analogue differs from Hoechst 33258 only in the location of its meta rather than para phenolic hydroxyl group and was designed to introduce the possibility of intermolecular hydrogen bonding to DNA via the phenol. Complex formation was shown to be 1:1 at 25 degrees C in phosphate buffer in D2O by 1D NMR spectroscopic titration of a solution of d(CGCGAATTCGCG)2 with meta-Hoechst. From 1D NMR spectroscopy the observed perturbations of the assigned chemical shifts of the oligonucleotide observed on binding meta-Hoechst could be used to locate the ligand in the central AATT stretch. By means of 2D NMR spectroscopic techniques, over 400 proton-proton NOEs were defined within the complex. DNA nonexchangeable resonance assignments were made using the sequential assignment method and NOESY. Binding the unsymmetrical ligand lifted the C2v symmetry of the DNA. Exchangeable hydrogens were assigned from NOESY data acquired in 85% H2O/15% D2O medium for the complex and showed differences between the Hoechst 33258 and meta-Hoechst complexes with d(CGCGAATTCGCG)2. The location of meta-Hoechst in the minor-groove AATT region was triangulated using 32 intermolecular NOEs determined for the complex. From the intermolecular NOEs involving the aromatic C-H protons of the phenolic ring of meta-Hoechst, it was clear that this region of the molecule did not rotate freely within the minor groove on the NMR time scale and was oriented with its hydroxyl group toward the floor of the minor groove, in line with the occurrence of the predicted hydrogen bonding between it and the DNA. The pKa of the N3H proton of meta-Hoechst in its bound state in this complex was measured as 6.1 by NMR spectroscopy, a value slightly elevated relative to estimates (approximately 5.2) of the pKa of this proton for the free ligand. Molecular mechanics and the distance restraints provided by the intermolecular NOEs were used in molecular modeling of the meta-Hoechst/d(CGCGAATTCGCG)2 complex, and the distances in the model were consistent with the formation of hydrogen bonds involving the m-OH group of meta-Hoechst and the DNA.

Mechanism of adenylate kinase. 1H, 13C, and 15N NMR assignments, secondary structures, and substrate binding sites.

Backbone 1H, 13C, and 15N NMR assignments were obtained for the complex of chicken muscle adenylate kinase (AK) with its bisubstrate analog, MgAP5A [magnesium P1,P5-bis(5'-adenosyl)-pentaphosphate]. The assignments were used to elucidate the secondary structures and the enzyme-MgAP5A interactions. The work involves two unusual features: the molecular weight of AK (21.6 kDa) is one of the largest, on a monomeric basis, for which nearly complete assignment has been reported to date, and the assignment was performed at pH 7.1 instead of the acidic pH used for most other proteins. The results are summarized as follows. Firstly, unambiguous sequential assignments of backbone resonances have been achieved effectively by the combined use of two sequential assignment methods: NOE-directed assignments and the recently developed 1J-coupling-directed assignments. The starting points of the assignments were provided by several specifically labeled enzyme samples. Over 90% of the backbone 1H, 13C, and 15N resonances have been assigned. Secondly, spin system information was obtained from the HCCH-TOCSY and HCCH-COSY experiments as well as from 2D homonuclear NMR data. Overall, the side-chain resonances of ca. 40% of the residues, including most of the those displaying NOEs with the adenosine moieties of MgAP5A, have been assigned. Thirdly, secondary structural elements in the AK-MgAP5A complex were identified by extensive analyses of 1H-15N 2D HMQC-NOESY and 3D NOESY-HMQC spectra. Overall, the enzyme consists of ca. 60% alpha-helices and a five-stranded parallel beta-sheet. The results are compared with the secondary structure of the free AK from porcine muscle in crystals [Dreusicke, D., Karplus, P. A., & Schulz, G. E. (1988) J. Mol. Biol. 199, 359-371]. Lastly, most of the intermolecular NOEs between AK and the adenosine moieties of MgAP5A have been identified: Thr39, Leu43, Gly64, Leu66, Val67, Val72, and Gln101 are in proximity to the adenosine moiety of the adenosine 5'-monophosphate site, whereas Thr23 is in proximity to that of the adenosine 5'-triphosphate site. These data are discussed in relation to previous results from site-directed mutagenesis, NMR, and X-ray studies and in relation to the mechanism of catalysis.

Structural study of a cell-wall mannan of Saccharomyces kluyveri IFO 1685 strain. Presence of a branched side chain and beta-1,2-linkage.

Acetolysis of the cell-wall mannan of Saccharomyces kluyveri under mild conditions, gave fragments with 1-6 mannose residues. The structures of mannopentaose and mannohexaose were determined to be [Formula; see text] respectively, by two-dimensional homonuclear Hartmann-Hahn spectroscopy and a sequential NMR assignment method that combines 1H-13C correlated spectroscopy, relayed coherence transfer spectroscopy, 1H-detected heteronuclear multiple-bond connectivity and methylation analysis. The H1 proton chemical shift of a neighboring alpha-1,2-linked mannose unit of the 3-O-substituted structure was shifted upfield by the addition of a mannose unit to the adjacent 3-O-substituted unit by an alpha-1,6 linkage. The characteristic H1--H2-correlated cross-peak of the alpha-1,3-linked mannose unit substituted by a beta-1,2 linkage, beta 1-->2Man alpha 1-->3, in the mannan of S. kluyveri, as also found by two-dimensional homonuclear Hartmann-Hahn spectroscopy in the mannan of Candida guilliermondii, a pathogenic yeast in man.