Proton n.m.r. of ferredoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.     

C solution state and solid state n.m.r. of wheat gluten

¹³C solid state and solution state, high resolution n.m.r. spectra have been used to characterize wheat gluten both as dry solid and hydrated mass. Most peaks in the spectra have been satisfactorily assigned. Four different environments within the material have been distinguished corresponding to rigid immobile protein, mobile hydrophobic side chains, mobile lipid and a mobile protein fraction apparent in the hydrated material.     

Structural versatility of peptides containing C-dialkylated glycines: conformational energy computations, i.r. absorption and H n.m.r. analysis of 1-aminocyclopropane-1-carboxylic acid homopeptides

Conformational energy computations on the 1-aminocyclopropane-1-carboxylic acid mono-, di-, and tripeptide amides, (Ac-(Ac₃c)_n---NHMe (n=1−3), indicate that this C^,-dialkylated, cyclic -amino acid residue is conformally restricted and that type-I(I′) β-bends and distorted 3₁₀-helices are particularly stable conformations for the di- and tripeptide amides, respectively. The results of the theoretical analysis are in agreement with those obtained in an i.r. absorption and ¹H n.m.r. investigation in chloroform solution of A.c.₃c-rich tri- and tetrapeptide esters. A comparisons is also made with the conclusions extracted from our previous work on peptides rich in Aib (-aminoisobutyric acid), Ac₅c(1-aminocyclopentane-1-carboxylic acid), and Ac₆c (1-aminocyclohexane-1-carboxylic acid).     

Effects of N-terminal extension peptides on the structure and stability of bovine pancreatic trypsin inhibitor studied by H n.m.r.

Four N-terminal extended species of the wild-type bovine pancreatic trypsin inhibitor (WT-BPTI), Arg-BPTI (1-BPTI), Met-Glu-Ala-Glu-BPTI (4-BPTI), Ser-Ile-Glu-Gly-Arg-BPTI (5-BPTI) and Gly-Ser-Ile-Glu-Gly-Arg-BPTI (6-BPTI) have been studied by 1H n.m.r. The overall structure of the protein is largely unaffected by the addition of extension peptides. pH titration effects on the C-terminal Ala 58 H beta chemical shift indicate that the structure of 1-BPTI at neutral pH is very similar to that of the WT protein, with a salt bridge between the main chain terminal charges. A salt bridge interaction is prevented by addition of the longer extension peptides. Temperature stabilities are measured by high temperature hydrogen isotope exchange and by microcalorimetry. The stability of 1-BPTI is equal to that of WT-BPTI. A slight decrease in stability is observed for longer extensions, following the order WT-BPTI = 1-BPTI < 5-BPTI = 6-BPTI < 4-BPTI. Small changes in chemical shift are observed for 30 invariant resonances in 4-, 5- and 6-BPTI and for a subset of this group in 1-BPTI. These protons are distributed over about half of the BPTI molecule. The size of the chemical shift changes for many resonances follow the same ranking as the temperature stability. The chemical shift effects are attributed to charge and dielectric effects from extension peptides that probably share a common orientation on the surface of BPTI.     

Proton n.m.r. of ferrodoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.     

C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

¹³C nuclear magnetic resonance spectroscopy (¹³C n.m.r.) was used to determine the pH titration parameters for the N-terminal N,N-[¹³C]dimethylamino and N,N-[¹³C]monomethylamino groups of glycophorins A^M and A^N, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin A^M.     

Interaction of group I cations with iota,kappa and lambda carrageenans studied by multinuclear n.m.r.

Results are reported for ²³Na, ³⁹K, ⁸⁷Rb and ¹³³Cs n.m.r. on kappa and iota carrageenans over a range of temperatures. Results for ²³Na and ³⁹K n.m.r. for lambda carrageenan are also given over the same range of temperatures. A variety of behaviour is observed which, in general, does not correlate with the rheological behaviour in these systems. It is concluded that non-specific ion interactions are of importance in determining rheological behaviour.     

400 MHz 1H n.m.r. study of the conformation and self-association of peptidolipin NA

Peptidolipin NA is a lipocyclopeptide extracted from Nocardia Asteroides having the formula:

Its conformation and self-association properties and those of its l-Val(6) analogue have been investigated in three solvents of different polarities: chloroform, pyridine and dimethylsulphoxide, using 400 MHz ¹H n.m.r. A model of the conformation of peptidolipin NA in pyridine has been proposed in which the peptide backbone is folded into a γ-turn around the l-Pro residue. Conformational changes are caused by l-Ala(6) → l-Val(6) replacement and by changing the solvent polarity. Nevertheless, the general shape of the peptide ring seems to be maintained. In chloroform, self-association occurs involving the (2) and, to a lesser extent, the (6) residues. Peptidolipin NA could be representative of natural amphiphiles in which a long hydrophobic tail is bound to a polar peptide moiety.     

Room temperature, low-field C-n.m.r. spectra of degraded carrageenans. Part II. On the specificity of the autohydrolysis reaction in kappa/iota and mu/nu structures

A kappa/iota carrageenan from Gigartina skottsbergii and a partially cyclized mu/nu carrageenan from Iridaea undulosa were submitted to autohydrolysis. The ¹³C-n.m.r. spectra of the degraded products give structural information on the polysaccharides and show clearly that besides the known splitting of 3,6-anhydrogalactosidic linkages, the linkage between - -galactose 2,6-disulphate and β- -galactose 4-sulphate is cleaved with similar specific reaction rate.     

C-n.m.r. spectral study of C reductively methylated glycopeptides derived from glycophorin A

¹³C-n.m.r. spectral data for ¹³C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the ¹³C-n.m.r. spectral data for the ¹³C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins A^M and A^N at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin A^M.     

13C magnetic resonance evidence for anisotropic molecular motion in collagen fibrils

Relaxation times and integrated intensities have been obtained from dipolar decoupled ¹³C magnetic resonance spectra of reconstituted fibrils of chick calvaria collagen enriched at the glycine C^a and C′ positions. The data obtained are consistent with a model in which collagen molecules reorient about the long axis of the helix with a rotational diffusion constant (R₁) of ~10⁷ s^?1, a value similar to that expected for the helix in solution. Data obtained from natural abundance ¹³C spectra of native (crosslinked) calf achilles tendon and rat tail tendon provide evidence of rapid anisotropic reorientation for at least 75% of the carbons in these tissues. Hence, our preliminary data indicate that, in these materials, the intermolecular interactions in the fibrilar collagen lattice can accommodate rapid reorientation at a majority of carbon sites.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Glucagon bound to perdeuterated dodecylphosphocholine micelles

The assignment of the ¹H nuclear magnetic resonance spectrum of glucagon bound to perdeuterated dodecylphosphocholine micelles with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 360 MHz is described. Sequential resonance assignments were obtained for all backbone and C^β protons except the N-terminal amino group and the amide proton of Ser2. The assignments of the non-labile amino acid side-chain protons are complete except for the γ-methylene protons of Gln20 and Gln24. These assignments provide a basis for the determination of the three-dimensional structure of lipid-bound glucagon.     

Agamococcidiorida ord. n. and Rhytidocystidae fam. n. for the Coccidian Genus Rhytidocystis Henneguy, 1907

Agamococcidiorida ord. n. and Rhytidocystidae fam. n. are established in the apicomplexan subclass Coccidiasina for the genus Rhytidocystis Henneguy, 1907. Dehornia Porchet-Henneré, 1972 is synonymized with Rhytidocystis.     

Conformation of reduced glutathione in aqueous solution by 1H and 13C n.m.r

We report on the conformation of reduced glutathione in solutions at low and physiological pH, examined with 1H and 13C n.m.r. spectroscopy. The tripeptide in 1H2O was shown to interconvert rapidly between an array of conformers; in addition, the carbon backbone of the glutamyl was more rigid than anticipated if the residue were freely mobile. This restricted motion results from interaction of the alpha-amino and alpha-carboxyl groups on the glutamyl, with the gamma-Glu-Cys peptide-carbonyl and amino, respectively. Our results support theoretical predictions of the conformation but they are at variance with previous ultraviolet spectroscopic and lower field n.m.r. studies.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor

The assignment of the ¹H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 500 MHz is described. The assignments are based entirely on the known amino acid sequence and the nuclear magnetic resonance data. Individual resonance assignments were obtained for all backbone and C^β protons, with the exception of those of Arg1, Pro2, Pro13 and the amide proton of Gly37. The side-chain resonance assignments are complete, with the exception of Pro2 and Pro13, the N^δ protons of Asn44 and the peripheral protons of the lysine residues and all but two of the arginine residues.     

Morphological conservatism in the foreleg structure of cicada hatchlings,Burmacicada protera n. gen., n. sp. in Burmese amber,Dominicicada youngi n. gen., n. sp. in Dominican amber and the extant Magicicada septendecim (L.) (Hemiptera: Cicadidae)

Two cicada hatchlings (Hemiptera: Cicadidae) in Burmese and Dominican amber are described as Burmacicada protera n. gen., n. sp. and Dominicicada youngi n. gen., n. sp., respectively. Although very similar in appearance, the two species can be separated by body contour, the nature of the process on the terminal antennomere and the shape and size of protrusions, teeth and spines on the forelegs. A comparison of the forelegs of the fossil hatchlings with those of an extant hatchling of the periodical cicada, Magicicada septendecim (L.), reveals a remarkable degree of morphological conservatism over 100 million years. A brief review of fossil cicadas is presented.     

Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence.

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotide sequences of the E. coli and B. brevis 16S rRNA chains, and of RNAse T1 oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzoic acid and C by bisulfite in naked 16S rRNA, and G by kethoxal in active and inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and T1. These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which 46 percent of the nucleotides of 16S rRNA are involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure.     

Bacteriophage degradation of Klebsiella K44 polysaccharide: an n.m.r. study and chemical proof of the position of the acetate group

Bacteriophages (phi) have been used to degrade polysaccharides into oligosaccharides containing one or more of their repeating units. The capsular polysaccharide from Klebsiella K44 contains an acetate group, and n.m.r. spectroscopy and chemical methods have been employed to prove its linkage to O-6 of the 4-linked glucose residue. Phage phi 44 was shown to be an alpha-glucosidase not influenced by the acetate moiety and thus able to depolymerize the polysaccharide into pentasaccharide repeating units, some of which contained acetate on O-6 of the reducing glucose residue. The two oligosaccharides were studied by 1H- and 13C-n.m.r. spectroscopy, and their spectra were compared with those of the native and the deacetylated polysaccharide. 13C-n.m.r. was a useful tool for locating the 6-linked acetate, the position of which was confirmed by the method of temporary protection using methyl vinyl ether. The importance of using bacteriophages to obtain oligosaccharides is highlighted by the better results obtained with the oligosaccharide in comparison to the polysaccharide, both in n.m.r. spectroscopy and the temporary protection method.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. II. Polarization of histidine 57 by substrate analogues and competitive inhibitors

The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin A_δ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to ?15.9, ?16.3 and ?17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of ?18.0 parts per million and the high pH position of ?14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN′ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-l-tryptophan to chymotrypsin A_δ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond.The His-Asp proton resonance was also monitored in complexes of chymotrypsin A_δ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of ?14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH.     

Predatory behaviour of the social orb-weaver spider,Geratonephila burmanica n. gen., n. sp. (Araneae: Nephilidae) with its wasp prey,Cascoscelio incassus n. gen., n. sp. (Hymenoptera: Platygastridae) in Early Cretaceous Burmese amber

The present work shows predatory behaviour of the social orb-weaver spider, Geratonephila burmanica n. gen., n. sp. (Araneae: Nephilidae) against a parasitic wasp, Cascoscelio incassus n. gen., n. sp. (Hymenoptera: Platygastridae) in Early Cretaceous Burmese amber. An adult male and juvenile of G. burmanica in the same web provide the first fossil evidence of sociality in spiders. The spider is characterised by a pedipalp with a hemispherical tegulum, a subtegulum curved at 180°and an apical spiralled embolas-conductor bent approximately 45°at midpoint. The male wasp is characterised by an ocellar tubercle, 12-segmented antennae with a feeble five-segmented clava, thick sensilla trichodea curvata with rounded ends on the claval antennomeres, a short uncus, a short post-marginal vein and a nebulose radial sector (Rs) vein extending from the uncus to the costal margin of the forewing. This is the first fossil evidence of spider sociality and a fossil spider attacking prey trapped in its web.