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.     

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.     

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.     

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.     

Minimised atomistic model and main evolution path for dominant B m I n clusters in boron diffusion

In this paper, we report our study on the minimised atomistic model (MAM) and the determination of an evolution path for dominant B _m I _n clusters during boron diffusion in kinetic Monte Carlo (KMC). It has been known that clusters generated after ion implantation play a decisive role in the enhanced boron diffusion at the tail region while being immobile at the peak region. Our MAM, based on the simple continuum model and the simple atomistic model, takes the smallest number of intermediate clusters into account as well as dominant clusters for the evolution path of interstitial clusters during boron diffusion. We find that intermediate clusters such as B₂I₃ and B₃I₃ play a significant role during the evolution of clusters despite the fact that the lifetimes of the corresponding intermediate clusters are relatively short due to low binding energies. Also, through our simulation results, we find the main evolution path of dominant clusters from B₂I to B₃I during thermal annealing in the MAM. Furthermore, our investigation reveals that the density of BI₂ clusters increases at the beginning of the annealing process while the density of B₃I increases at a later stage. KMC simulation results are compared with experimental SIMS data, which support our theoretical model.     

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.     

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.     

Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra: Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations

Two different, theoretical studies of intramolecular proton-proton distances in polypeptide chains are described. Firstly, the distances between amide, C^α and C^β protons of neighbouring residues in the amino acid sequence, which correspond to the sterically allowed values for the dihedral angles φ_i, ψ_i and χ_i¹, were computed. Secondly, the frequency with which short distances occur between amide, C^α and C^β protons of neighbouring and distant residues in the amino acid sequence were statistically evaluated in a representative sample of globular protein crystal structures. Both approaches imply that semi-quantitative measurements of short, non-bonding proton-proton distances, e.g. by nuclear Overhauser experiments, should present a reliable and generally applicable method for sequential, individual resonance assignments in protein ¹H nuclear magnetic resonance spectra. Similar calculations imply that corresponding distance measurements can be used for resonance assignments in the side-chains of the aromatic amino acid residues, asparagine and glutamine, where the complete spin systems cannot usually be identified from through-bond spin-spin coupling connectivities.     

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.     

Detection of secondary structure in glycosaminoglycans via the H n.m.r. signal of the acetamido NH group.

Two simple methods for dissolving salts of acid glycosaminoglycans with inorganic cations (e.g. Li+ and Na+) in dry dimethyl sulphoxide are described. Complete n.m.r. spectra of, e.g., Na+ and Li+ salts of chondroitin sulphate and keratan sulphate were obtained on these solutions. In [2H6]dimethyl sulphoxide the NH resonance of 2-acetamido-2-deoxy hexosides is in the range 7.2-8.0 delta, but is downfield (8.3-9.3 delta) when the NH is H-bonded to -CO2-. Heparan sulphate shows two NH resonances, of which one (at 8.3 delta) is probably indicative of H-bonding. Space-filling models show that a very close approach of NH to -CO2- across the alpha-glucosaminidic bond is possible, and a solution configuration for heparan sulphate is proposed. The n.m.r. results are entirely compatible with interpretations of periodate-oxidation kinetics, based on H-bonded secondary structures present in hyaluronate and chondroitin sulphates, but not in dermatan (or keratan) sulphate.     

Sequence-specific 1H n.m.r. assignments and determination of the three-dimensional structure of reduced Escherichia coli glutaredoxin

The determination of the nuclear magnetic resonance structure of reduced E. coli glutaredoxin in aqueous solution is described. Based on nearly complete, sequence-specific resonance assignments, 813 nuclear Overhauser effect distance constraints and 191 dihedral angle constraints were employed as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of reduced glutaredoxin is made up of three helices and four-stranded beta-sheet. The first strand of the beta-sheet (residues 2 to 7) runs parallel to the second strand (32 to 37) and antiparallel to the third strand (61 to 64), and the sheet is extended in an antiparallel fashion with a fourth strand (67 to 69). The first helix with residues 13 to 28 and the last helix (71 to 83) run parallel to each other on one side of the beta-sheet, with their direction opposite to that of the two parallel beta-strands, and the helix formed by residues 44 to 53 fills space available due to the twist of the beta-sheet and the reduced length of the last two beta-strands. The active site Cys11-Pro-Tyr-Cys14 is located after the first beta-strand and occupies the latter part of the loop connecting this strand with the first helix.     

1H and 13C n.m.r. studies of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin

1H and 13C n.m.r. study of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, obtained by replacing each peptide bond by a "reduced peptide bond", one at a time, e.g. Boc-Trp psi (CH2NH)Leu-Asp-Phe-NH2 2, Boc-Trp-Leu psi (CH2NH) Asp-Phe-NH2 3, Boc-Trp-Leu-Asp psi (CH2NH)Phe-NH2 4, were reported. The CH2NH bond was completely characterized. 1H and 13C spectroscopic data were reported. It appeared from the present work that the modifications produced by the replacement of a peptide bond by a CH2NH bond were localized around the CH2NH.     

1H, 13C and 31P n.m.r. spectra and molecular conformation of myo-inositol 1,4,5-triphosphate.

Multinuclear n.m.r. spectroscopy has been used to confirm the identify of inositol 1,4,5-trisphosphate and to deduce its molecular conformation as having five equatorial and one axial ring substituents.