Quantitative analysis of protein far UV circular dichroism spectra by neural networks

A new method based on neural network theory is presented toanalyze and quantify the information content of far UV circulardichroism spectra. Using a backpropagation network model witha single hidden layer between input and output, it was possibleto deduce five different secondary structure fractions (helix,parallel and antiparallel ß-sheet, ß-turnand random coil) with satisfactory correlations between calculatedand measured secondary structure data. We demonstrate that foreach wavelength interval a specific network is suitable. Theremaining discrepancy between the secondary structure data fromneural network prediction and crystallography may be attributedto errors in the determination of protein concentration andrandom noise in the CD signal, as indicated by simulations.     

Nuclear magnetic resonance studies of isolated structural domains of yeast phosphoglycerate kinase

The structural integrity and substrate binding properties ofthe two genetically engineered domains of yeast phosphoglyceratekinase were investigated using one- and two-dimensional nuclearmagnetic resonance techniques. Both domains were found to foldwith regions of native-like structure, with the N-domain showinggreater conformational flexibility than the C-domain. The ‘basicpatch’ region of the N-domain is, however, clearly perturbedby removal of the C-domain. This is most likely due to the absenceof stabilizing interactions between the C-terminal peptide (including-helices XIII and XIV) and the N-domain. The C-domain is ableto bind nucleotide with an affinity only three times less thanthat of the native protein.     

Solution structure of human corticotropin releasing factor by 1H NMR and distance geometry with restrained molecular dynamics

The structure of human corticotropin releasing factor (hCRF)has been determined by proton nuclear magnetic resonance (¹HNMR) in a mixed-solvent system of 66% trifluoroethanol/34% H₂Oat pH 3.8 and 37°C. Nearly complete resonance assignmentwas achieved by using standard two-dimensional methods. Distancerestraints for structure calculations were obtained by qualitativeanalysis of intra- and interresidue nuclear Overhauser effects.Structures were obtained from the distance restraints by distancegeometry, followed by refinement using molecular dynamics andwere completed with amide hydrogen exchange data. The structureof hCRF in this solvent comprises an extended N-terminal tetrapeptideconnected to a well-defined -helix between residues 6 and 36.The first half of the -helix (residues 6–20) is clearlyamphipathic. The five carboxy-terminal residues are predominantlydisordered.     

Solution conformations of human growth hormone releasing factor: comparison of the restrained molecular dynamics and distance geometry methods for a system without long-range distance data

A series of three-dimensional structures of the 1–29 fragmentof human growth hormone releasing factor in trifluoroethanolhave been determined by molecular dynamics and distance geometrymethods. The resulting structures satisfy information from nuclearOverhauser effect (NOE) distance data and an empirical potentialenergy function. Although the polypeptide was found to havean ordered structure in all simulations, the NOE data were notsufficient for global convergence to a unique three-dimensionalgeometry. Several satisfactory structures have been determined,all of which are extended conformations consisting of a shortß-strand and two -helices (residues 6–13 andresidues 16–29) connected by short segments of less welldefined secondary structure. Because of the lack of NOE dataconnecting the helix segments, their relative orientation isnot uniquely determined.     

核磁共振法测定纤维含油率

介绍了一种全新的化纤含油率检测方法——核磁共振法(NMR),并在长丝和无硅中空纤维含油率测试中对NMR法和传统方法进行了比较。     

Nuclear magnetic resonance approach to the characterization of siloxane-silica network-like structures

This work deals with mesh size properties of network-like structures associated with random siloxane-silica mixtures. The transverse magnetic relaxation function of protons is shown to obey a superposition property controlled by the silica concentration C_Si, in the range 0C_Si0.50 (w/w). Correspondingly, its invariant mathematical structure is governed by the average residual energy of dipole-dipole interactions only. The parameter Δ_e is given the role of a timescale shift factor; it is related to the average mesh size of a given network. Nuclear magnetic resonance measurements show that silica aggregates do not induce any strong deviation from the primary entanglement system established in a pure siloxane melt. Whether infinite siloxane-silica clusters are swollen by polymer chains or whether finite size clusters are diluted in a siloxane melt, the resulting networks are described by similar distribution functions of elementary chains connecting neighbouring entanglements; these functions are shifted towards smaller mesh sizes upon addition of silica. These properties hold for several high molecular weight samples: , 2.4 × 10⁵ and 1.6 × 10⁵, respectively.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues20–83) of barley serine proteinase inhibitor 2 (BSPI-2)is determined on the basis of 403 interproton distance, 34 øbackbone torsion angle and 26 hydrogen bonding restraints derivedfrom n.m.r. measurements. A total of 11 converged structureswere computed using a metric matrix distance geometry algorithmand refined by restrained molecular dynamics. The average rmsdifference between the final 11 structures and the mean structureobtained by averaging their coordinates is 1.4±0.2 Åfor the backbone atoms and 2.1±0.1 A for all atoms. Theoverall structure, which is almost identical to that found byX-ray crystallography, is disc shaped and consists of a centralfour component mixed parallel and antiparallel ß-sheetflanked by a 13 residue helix on one side and the reactivesite loop on the other.     

Primary and predicted secondary structures of the caseins in relation to their biological functions

In free solution, the caseins behave as non-compact and largelyflexible molecules with a high proportion of residues accessibleto solvent. Historically, they have been described as randomcoil-type proteins with only a nutritional function. Nevertheless,secondary structure prediction algorithms indicate that manyparts of the (unphosphorylated, unglyco-sylated) polypeptidechains can form regular structures. In particular, a recurrentmotif of the Ca²⁺-sensitive caseins in man, rat, mouse, guineapig and ruminant species is an a-helix-loop-a-helix conformationin which the loop region typically contains a cluster of sitesof phosphorylation. The biological function of the caseins isconsidered and it is suggested that the potential or actualconformations of the group of Ca²⁺-sensitive caseins are suitedto the function of modulating the precipitation of calcium phosphatefrom solution. Either they can act as sites for nucleation orthey can bind rapidly to calcium phosphate nuclei as they formspontaneously from supersaturated solution.     

A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy

Restriction enzymes (REases) are commercial reagents commonly used in DNA manipulations and mapping. They are regarded as very attractive models for studying protein-DNA interactions and valuable targets for protein engineering. Their amino acid sequences usually show no similarities to other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. Hence, they are extremely hard targets for structure prediction and modeling. NlaIV is a Type II REase, which recognizes the interrupted palindromic sequence GGNNCC (where N indicates any base) and cleaves it in the middle, leaving blunt ends. NlaIV shows no sequence similarity to other proteins and virtually nothing is known about its sequence-structure-function relationships. Using protein fold recognition, we identified a remote relationship between NlaIV and EcoRV, an extensively studied REase, which recognizes the GATATC sequence and whose crystal structure has been determined. Using the 'FRankenstein's monster' approach we constructed a comparative model of NlaIV based on the EcoRV template and used it to predict the catalytic and DNA-binding residues. The model was validated by site-directed mutagenesis and analysis of the activity of the mutants in vivo and in vitro as well as structural characterization of the wild-type enzyme and two mutants by circular dichroism spectroscopy. The structural model of the NlaIV-DNA complex suggests regions of the protein sequence that may interact with the 'non-specific' bases of the target and thus it provides insight into the evolution of sequence specificity in restriction enzymes and may help engineer REases with novel specificities. Before this analysis was carried out, neither the three-dimensional fold of NlaIV, its evolutionary relationships or its catalytic or DNA-binding residues were known. Hence our analysis may be regarded as a paradigm for studies aiming at reducing 'white spaces' on the evolutionary landscape of sequence-function relationships by combining bioinformatics with simple experimental assays.     

Similarity graphing and enzyme-reaction database: methods to detect sequence regions of importance for recognition of chemical structures

We developed a new method which searches sequence segments responsiblefor the recognition of a given chemical structure. These segmentsare detected as those locally conserved among a sequence tobe analyzed (target sequence) and a set of sequences (referencesequences). Reference sequences are the sequences of functionallyrelated proteins, ligands of which contain a common chemicalsubstructure in their molecular structures. ‘Similaritygraphing’ cuts target sequences into segments, alignsthem with reference sequence pairwise, calculates the degreeof similarity for each alignment, and shows graphically cumulativesimilarity values on target sequence. Any locally conservedregions, short or long in length and weak or strong in similarity,are detected at their optimal conditions by adjusting threeparameters. The ‘enzyme-reaction database’ containschemical structures and their related enzymes. When a chemicalsubstructure is input into the database, sequences of the enzymesrelated to the input substructure are systematically searchedfrom the NBRF sequence database and output as reference sequences.Examples of analysis using similarity graphing in combinationwith the enzyme-reaction database showed a great potentialityin the systematic analysis of the relationships between sequencesand molecular recognitions for protein engineering.     

Detection of secondary structure elements in proteins by hydrophobic cluster analysis

Hydrophobic cluster analysis (HCA) is a protein sequence comparisonmethod based on -helical representations of the sequences wherethe size, shape and orientation of the clusters of hydrophobicresidues are primarily compared. The effectiveness of HCA hasbeen suggested to originate from its potential ability to focuson the residues forming the hydrophobic core of globular proteins.We have addressed the robustness of the bidimensional representationused for HCA in its ability to detect the regular secondarystructure elements of proteins. Various parameters have beenstudied such as those governing cluster size and limits, thehydrophobic residues constituting the clusters as well as thepotential shift of the cluster positions with respect to theposition of the regular secondary structure elements. The followingresults have been found to support the -helical bidimensionalrepresentation used in HCA: (i) there is a positive correlation(clearly above background noise) between the hydrophobic clustersand the regular secondary structure elements in proteins; (ii)the hydrophobic clusters are centred on the regular secondarystructure elements; (iii) the pitch of the helical representationwhich gives the best correspondence is that of an -helix. Thecorrespondence between hydrophobic clusters and regular secondarystructure elements suggests a way to implement variable gappenalties during the automatic alignment of protein sequences.     

红外和核磁共振法鉴定聚醚型聚酯的组成结构

利用红外吸收光谱及核磁共振波谱仪的一维和二维技术对一种未知聚合物试样进行鉴定。由核磁共振二维技术分析得出聚醚酯的序列结构及其分布。结果表明:该未知聚合物试样为聚醚酯弹性体,由对苯二甲酸、间苯二甲酸、新戊二醇、乙二醇、聚四氢呋喃醚共聚而成。     

Nuclear magnetic resonance studies and molecular dynamics simulations of the solution conformation of a 'designed', {alpha}-helical peptide

The complete three-dimensional structure in methanol of an amphipathica-helical peptide, that has been designed by taking into accountthe three-dimensional structures of small haemolytic peptides,secondary structure prediction algorithms and the well documentedliterature on -helix stabilizing factors, has been elucidatedby two-dimensional NMR spectroscopy. Initially various two-dimensionalspectra (COSY, TOCSY, and NOESY) allowed the complete sequencespecific assignment of all signals in the ¹H spectrum. Consequentlytrial structures were generated which were then subjected tomolecular dynamics simulations using 121 NOE-derived distancesand 25 vicinal coupling constant values as structural restraintsto give a final set of calculated structures. These structuresare in complete agreement with the results of a circular dichroismstudy and reveal that the peptide adopted a highly ordered a-helicalconformation. Details of the structure which throw light onfuture peptide/protein design are discussed.     

Influence of secondary structure on the hydration of serine, threonine and tyrosine residues in proteins

Previous analysis of experimental data on the solvation of highresolution protein structures has shown that preferred interactionsites for water molecules exist around most amino acid sidechains. We have extended this analysis to look in more detailat the distributions around serine, threonine and tyrosine.We find that for serine and threonine side chains the preferredinteraction sites of solvent molecules with the hydroxyl groupdepends on secondary structure and the _X1 torsion angle of theside chain. For tyrosine side chains the hydroxyl group is toofar from the main chain to reflect secondary structure influences.Specific patterns of hydration are observed in which water molecules‘bridge’ between the hydroxyl side-chain atom andanother main chain or side-chain atom.     

Comparison of three algorithms for the assignment of secondary structure in proteins: the advantages of a consensus assignment

Accurate assignments of secondary structures in proteins arecrucial for a useful comparison with theoretical predictions.Three major programs which automatically determine the locationof helices and strands are used for this purpose, namely DSSP,P-Curve and Define. Their results have been compared for a non-redundantdatabase of 154 proteins. On a residue per residue basis, thepercentage match score is only 63% between the three methods.While these methods agree on the overall number of residuesin each of the three states (helix, strand or coil), they differon the number of helices or strands, thus implying a wide discrepancyin the length of assigned structural elements. Moreover, thelength distribution of helices and strands points to the existenceof artefacts inherent to each assignment algorithm. To overcomethese difficulties a consensus assignment is proposed whereeach residue is assigned to the state determined by at leasttwo of the three methods. With this assignment the artefactsof each algorithm are attenuated. The residues assigned in thesame state by the three methods are better predicted than theothers. This assignment will thus be useful for analysing thesuccess rate of prediction methods more accurately.     

Interaction of metal ions with carboxylic and carboxamide groups in protein structures

An analysis of the geometry of metal binding by carboxylic andcarboxamide groups in proteins is presented. Most of the ligandsare from aspartic and glutamic acid side chains. Water moleculesbound to carboxylate anions are known to interact with oxygenlone-pairs. However, metal ions are also found to approach thecarboxylate group along the C - O direction. More metal ionsare found to be along the syn than the anti lone-pair direction.This seems to be the result of the stability of the five-memberedring that is formed by the carboxylate anion hydrogen bondedto a ligand water molecule and the metal ion in the syn position.Ligand residues are usually from the helix, turn or regionswith no regular secondary structure. Because of the steric interactionsassociated with bringing all the ligands around a metal center,a calcium ion can bind only near the ends of a helix; a metal,like zinc, with a low coordination number, can bind anywherein the helix. Based on the analysis of the positions of watermolecules in the metal coordination sphere, the sequence ofthe EF hand (a calcium-binding structure) is discussed.     

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M·EcoRV) is an -adeninemethyltransferase. We have used two different programs to predictthe secondary structure of M·EcoRV. The resulting consensusprediction was tested by a mutant profiling analysis. 29 neutralmutations of M·EcoRV were generated by five cycles ofrandom mutagenesis and selection for active variants to increasethe reliability of the prediction and to get a secondary structureprediction for some ambiguously predicted regions. The predictedconsensus secondary structure elements could be aligned to thecommon topology of the structures of the catalytic domains ofM·HhaI and M·TaqI. In a complementary approachwe have isolated nine catalytically inactive single mutants.Five of these mutants contain an amino acid exchange withinthe catalytic domain of M·EcoRV (Val20-Ala, Lys81Arg,Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant bindsDNA similarly to wild-type M·EcoRV, but is catalyticallyinactive. Hence this mutant behaves like a bona fide activesite mutant. According to the structure prediction, Trp231 islocated in a loop at the putative active site of M·EcoRV.The other inactive mutants were insoluble. They contain aminoacid exchanges within the conserved amino acid motifs X, IIIor IV in M·EcoRV confirming the importance of these regions.     

用固体核磁技术分析PE 100管材料的抗熔垂性

用固体核磁技术分析了2种具有不同抗熔垂性能的聚乙烯管材料的凝聚态结构与流变性能。管材树脂的抗熔垂性能与其核磁迟豫特性紧密相关,好的抗熔垂性能对应更长的纵向迟豫时间。抗熔垂性能好的双峰管材料的重均分子量更高,高相对分子质量部分的含量更多。     

Enzyme-catalyzed polymerization of 8-hydroxyquinoline-5-sulfonate by in situ nuclear magnetic resonance spectroscopy

In this report, we describe the use of in situ NMR spectroscopy to elucidate the mechanism of horseradish peroxidase-catalyzed oxidative free-radical coupling of phenols. We demonstrate the potential of the technique for the polymerization of 8-hydroxyquinoline-5-sulfonate (HQS). Based on the structural changes, we establish the involvement of ortho- and para-position protons (to the hydroxyl group) in the oxidative free-radical coupling polymerization with their relative preferences. For example, in HQS, we establish that the positions 2, 4, and 7 are involved in the chemical bonding with the order of preference being 7 ≥ 2 > 4. Analyses of ¹³C-NMR data suggest the formation of C—C- and C—O—C-type coupling bonds during enzymatic polymerization. © 1998 John Wiley & Sons, Inc. J. Appl. Polym. Sci. 70: 1257–1264, 1998     

Protein fold recognition by threading: comparison of algorithms and analysis of results

Optimal sequence threading can be used to recognize membersof a library of protein folds which are closely related in 3-Dstructure to the native fold of an input test sequence, evenwhen the test sequence is not significantly homologous to thesequence of any member of the fold library. The methods providean alignment between the residues of the test sequence and theresidue positions in a template fold. This alignment optimizesa score function, and the predicted fold is the highest scoringmember of the library of folds. Most score functions containa pairwise interaction energy term. This, coupled with the needto introduce gaps into the alignment, means that the optimizationproblem is NP hard. We report a comparison between two heuristicoptimization algorithms used in the literature, double dynamicprogramming and an iterative algorithm based on the so-calledfrozen approximation. These are compared in terms of both theranking of likely folds and the quality of the alignment produced.