Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability

We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1. We utilize these Ala-WT DeltaDeltaG values to filter previously described Pro-WT DeltaDeltaG values, creating Pro-Ala DeltaDeltaG values that specifically assess the sensitivity of a sequence position, in the structural context of the Abeta fibril, to replacement by proline. The results provide a conservative view of the energetics of Abeta(1-40) fibril structure, indicating that positions 18-21, 25-26, and 32-33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14-17, 22, 24, 27-31, and 34-39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Abeta(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 degrees C under unstirred ("quiescent") conditions.     

Solution structure of neuromedin B by (1)H nuclear magnetic resonance spectroscopy.

The solution structure of neuromedin B (NMB) was investigated using two-dimensional nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking environments. NMB adopts a relaxed helical conformation from Trp(4) to Met(10) in 50% aqueous 2,2, 2-trifluoroethanol (TFE) solution and in 150 mM SDS micelles. Sidechain atoms of the three residues, Trp(4), His(8) and Phe(9) orient toward the same direction and these residues might play a key role on interacting with hydrophobic acyl chains of the phospholipids in the membrane. NOESY experiments performed on NMB in non-deuterated SDS micelle show that aromatic ring protons of Trp(4) and Phe(9) residues are in close contact with methylene protons of SDS micelles. In addition, proton longitudinal relaxation data proved that the interactions between NMB with SDS micelle are characterized as extrinsic interaction. Trp(4) and Phe(9) seem to be important in interaction with receptor and this agrees with the previous studies of structure-activity relationship (Howell, D.C. et al. (1996) Int. J. Pept. Protein Res. 48, 522-531). These conformational features might be helpful in understanding the molecular mechanism of the function of NMB and developing the efficient drugs.     

The secondary structure in solution of acyl-coenzyme A binding protein from bovine liver using 1H nuclear magnetic resonance spectroscopy

Acyl-coenzyme A binding protein from bovine liver and the protein expressed in Escherichia coli by the recombinant gene of this protein have been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein has, in addition to the ability to bind acyl-coenzyme A, been reported to have several important physiological and biochemical functions. It is known as the diazepam binding inhibitor, as a putative neurotransmitter, as a regulator of insulin release from pancreatic cells, and as a mediator in corticotropin-dependent adrenal steroidogenesis. The only difference between the protein produced by recombinant techniques and the native acyl-coenzyme A binding protein is the N-terminal acetyl group present only in the native protein. The two proteins have 86 amino acid residues and a molecular mass of approximately 10,000 Da. Complete assignment of the 1H nuclear magnetic resonances has been obtained for a major proportion of the amino acid residues (55 residues), and partial assignment has been achieved for the others (31 residues). Sequential nuclear Overhauser effects have demonstrated that the protein has a secondary structure consisting of four alpha-helices of residues 1-15, 22-35, 52-60, and 68-85. Furthermore, a large number of long-range nuclear Overhauser effects have been identified, indicating that the assignment given here will provide a basis for a structure determination of this protein in solution by nuclear magnetic resonance spectroscopy.     

Mapping the core of the beta(2)-microglobulin amyloid fibril by H/D exchange

Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.     

Secondary structure in solution of barwin from barley seed using 1H nuclear magnetic resonance spectroscopy.

Barwin, a basic protein from barley seed of 125 amino acid residues, has been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein is closely related to the C-terminal domain of proteins whose synthesis is induced by wounding, the so-called win proteins. These proteins may, therefore, have a role in the defense against fungal attack. Full assignment of the 1H nuclear magnetic resonances has been obtained for 104 amino acid residues, and 18 amino acid spin systems were partially assigned. Sequence-specific assignment using nuclear Overhauser spectroscopy has been achieved for 122 of the 125 residues. This has revealed that the secondary structure of the protein is dominated by a large four-stranded antiparallel beta-sheet consisting of the strands Gln2-Thr9, Lys65-Asn71, Gln77-Arg81, and His113-Val121, a small parallel beta-sheet of the strands Trp48-Cys52 and Asp84-Ala87, which together account for a third of the protein. Sequential effects indicate the presence of three small alpha-helices, Tyr30-Lys38, Leu40-Tyr46, and Thr97-Asp103. The secondary structure in other regions of the sequence is characterized mainly by loops and turns and regions where no regular secondary structure arrangement could be identified. A large number of long-range nuclear Overhauser effects has been identified, and these have been used, together with sequential and intranuclear Overhauser effects, for a calculation of the protein's three-dimensional structure.     

Amyloid fibril dynamics revealed by combined hydrogen/deuterium exchange and nuclear magnetic resonance

A general method to explore the dynamic nature of amyloid fibrils is described, combining hydrogen/deuterium exchange and nuclear magnetic resonance spectroscopy to determine the exchange rates of individual amide protons within an amyloid fibril. Our method was applied to fibrils formed by the amyloid-β(1-40) peptide, the major protein component of amyloid plaques in Alzheimer’s disease. The fastest exchange rates were detected among the first 14 residues of the peptide, a stretch known to be poorly structured within the fibril. Considerably slower exchange rates were observed in the remainder of the peptide within the β-strand-turn-β-strand motif that constitutes the fibrillar core.     

Cu（Ⅱ） Potentiation of Alzheimer Aβ1-40 Cytotoxicity and Transition on its Secondary Structure

Mounting evidence has shown that dyshomeostasis of the redox-active biometals such as Cuand Fe can lead to oxidative stress,which plays a key role in the neuropathology of Alzheimer's disease(AD).Here we demonstrate that with the formation of Cu(Ⅱ)·Aβ1-40 complexes,copper markedly potentiatesthe neurotoxicity exhibited by β-amyloid peptide (Aβ).A greater amount of hydrogen peroxide was releasedwhen Cu(Ⅱ)·Aβ1-40 complexes was added to the xanthine oxidase/xanthine system detected by potassiumiodide spectrophotometry.Copper bound to Aβ1-40 was observed by electron paramagnetic resonance(EPR) spectroscopy.Circular dichroism (CD) studies indicated that copper chelation could cause a structuraltransition of Aβ.The addition of copper to Aβ introduced an increase on β-sheet as well as α-helix,whichmay be responsible for the aggregation of Aβ.We hypothesized that Aβ aggregation induced by copper maybe responsible for local injury in AD.The interaction between Cu~(2 ) and Aβ also provides a possible mechanismfor the enrichment of metal ions in amyloid plaques in the AD brain.     

Solution conformation of leiurotoxin I (scyllatoxin) by 1H nuclear magnetic resonance. Resonance assignment and secondary structure

Cells of Synechococcus 6301 were briefly exposed to a phycocyanin-absorbed light in the presence of DCMU. PS II trap closure was then estimated from fluorescence induction measurements with excitation light absorbed predominantly either by chlorophyll or by phycocyanin. In cells adapted to light-state 2, the exposure to light absorbed by phycocyanin closed only a proportion of the PS II centres that could be closed by exposure to light absorbed by chlorophyll. This distinction was reduced in cells adapted to light-state 1. We conclude that a proportion of PS II core complexes become decoupled from the phycobilisomes during the transition to light-state 2.     

1H nuclear magnetic resonance assignments and secondary structure of porcine C5ades Arg

Sequence-specific assignments of the 1H nuclear magnetic resonance spectrum of porcine C5ades Arg are described. Assignments were facilitated by comparison of spectra obtained in H2O with partially exchanged spectra obtained in 2H2O. The sequence-specific assignments thus obtained were used to characterized the regular secondary structure in the protein, which is helical in the regions 2 to 11, 16 to 27, 35 to 41 and 45 to 64. The structure is very similar to that of human and bovine C5a.     

Determination of the secondary structure of interleukin-8 by nuclear magnetic resonance spectroscopy

The solution conformation of interleukin-8 (IL-8), a small protein of 72 residues with a wide range of proinflammatory activities, has been investigated by two-dimensional NMR spectroscopy. The 1H-NMR spectrum of IL-8 is assigned in a sequential manner and regular elements of secondary structure are identified on the basis of a qualitative interpretation of the nuclear Overhauser, coupling constant and amide exchange data. The IL-8 monomer contains a triple stranded anti-parallel beta-sheet arranged in a Greek key and a long C-terminal helix (residues 57-72). It is shown that IL-8 is a dimer in solution in which the interface is principally formed by six backbone hydrogen bonds between residues 25, 27, and 29 of one monomer and residues 29, 27, and 25, respectively, of the other. As a result, the two units of the dimer form a contiguous six-stranded anti-parallel beta-sheet. The secondary structure of IL-8 is similar to that found in the crystal structure of the sequence related protein platelet factor 4.     

Spatial structure determination of antiarrhythmic peptide using nuclear magnetic resonance spectroscopy

The peptide AAP10 was synthesized according to the Merrifield technique following the Fmoc-strategy and its spatial structure in aqueous solution studied with NMR principles. It is known from previous studies that the peptide has antiarrhythmic activity and inhibits cardiac ischemia induced alterations of the activation pattern, decreases the activation–recovery interval (ARI) dispersion and improves cellular coupling via enhancement of gap junction conductance (2, 2, 3, 4). The peptide was synthesized as a peptide amide. Two different semi cyclic conformations were characterized.     

Omega 1-decoupled 1H homonuclear shift-correlated nuclear magnetic resonance spectroscopy (COSYDEC) applied to steroids

Problems of cross-peak overlap in two-dimensional 1H homonuclear shift-correlated (COSY) spectra of steroids can often be avoided by use of the omega 1-decoupled COSY (COSYDEC) method. The selection of experimental parameters is discussed, and COSYDEC spectra are illustrated for 17a-oxa-D-homoandrost-4-ene-3,17-dione (testololactone), testosterone, and 17 alpha-hydroxyprogesterone. In a good case, a COSYDEC spectrum obtained at 250 MHz allows cross-peak recognition and assignment with facility comparable to that available only at 500 MHz for normal COSY spectra.     

Leishmania donovani: Metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, -glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.     

Solution conformation of brazzein by H nuclear magnetic resonance: resonance assignment and secondary structure

Brazzein is a sweet-tasting protein isolated from the fruit of the West African plant Pentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far, it is also highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300K) is presented. Complete sequence specific assignment of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one -helix (residues 21–29), one short 3₁₀-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus.     

Solution conformation of leiurotoxin I (scyllatoxin) by H nuclear magnetic resonance Resonance assignment and secondary structure

A proton NMR study at 500 MHz of leiurotoxin I in water is presented. Nearly complete sequence-specific assignments of the individual backbone and side-chain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of this toxin is inferred from a combination of short-range nuclear Overhauser enhancements, scalar couplings and proton/deuteron exchange rates. Three disulflde bridges locate the N-terminal part (that is -helical from residue 6 to 16) on one side of a C-terminal two stranded antiparallel β sheet (from Leu¹⁸ to Val²⁹). The latter features a tight turn at Gly²³-Asp²⁴.     

Binding of an oxindole alkaloid from Uncaria tomentosa to amyloid protein (Abeta1-40)

The primary aim of this work was to determine the interactions of an oxindole alkaloid (mitraphylline) isolated from Uncaria tomentosa with beta-amyloid 1-40 (Abeta1-40 protein) applying the capillary electrophoresis (CE) method. Specifically the Hummel-Dreyer method and Scatchard analysis were performed to study the binding of oxindole alkaloids with Abeta1-40 protein. Prior to these studies extraction of the alkaloid of interest was carried out. Identification of the isolated alkaloid was performed by the use of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) combined with electrospray ionization mass spectrometry (ESI-MS). The proposed approach was proved to be an efficient and accurate method for specific compound isolation and identification purposes. Moreover, analytical information from the CE approach can be considered as the valuable tool for binding constant determination. The binding constant of mitraphylline with Abeta1-40 protein determined by the Hummel-Dreyer method and Scatchard analysis equals K = 9.95 x 10(5) M(-1). The results obtained showed the significant binding of the tested compound with Abeta1-40 protein. These results are discussed and interpreted in the view of developing a strategy for identification of novel compounds of great importance in Alzheimer disease therapy.     

Determining the critical nucleus and mechanism of fibril elongation of the Alzheimer's Abeta(1-40) peptide

We use a coarse-grained protein model to characterize the critical nucleus, structural stability, and fibril elongation propensity of Aβ_1-40 oligomers for the C_2x and C_2z quaternary forms proposed by solid-state NMR. By estimating equilibrium populations of structurally stable and unstable protofibrils, we determine the shift in the dominant population from free monomer to ordered fibril at a critical nucleus of ten chains for the C_2x and C_2z forms. We find that a minimum assembly of 16 monomer chains is necessary to mimic a mature fibril, and show that its structural stability correlates with a plateau in the hydrophobic residue density and a decrease in the likelihood of losing hydrophobic interactions by rotating the fibril subunits. While Aβ_1-40 protofibrils show similar structural stability for both C_2x and C_2z quaternary structures, we find that the fibril elongation propensity is greater for the C_2z form relative to the C_2x form. We attribute the increased propensity for elongation of the C_2z form as being due to a stagger in the interdigitation of the N-terminal and C-terminal β-strands, resulting in structural asymmetry in the presented fibril ends that decreases the amount of incorrect addition to the N terminus on one end. We show that because different combinations of stagger and quaternary structure affect the structural symmetry of the fibril end, we propose that differences in quaternary structures will affect directional growth patterns and possibly different morphologies in the mature fiber.     

Determination of the three-dimensional structure of iberiotoxin in solution by 1H nuclear magnetic resonance spectroscopy.

The solution structure of chemically synthesized iberiotoxin, a scorpion toxin that blocks Ca(2+)-activated K+ channels, has been determined using 2D 1H NMR spectroscopy. Analysis of the NOEs, coupling constants, and HN-DN exchange rates indicates the structure consists of an antiparallel beta-sheet from residues 25 to 36, with a type 1 turn at residues 30-31, and a helix from residues 13 to 21. The carboxyl-terminal residues form a short, and distorted, third strand of the sheet. The NMR data are consistent with disulfide bonds from residues 7 to 28, 13 to 33, and 17 to 35. The disulfide bridging presents the same profile as in other scorpion toxins, where a Cys-X-Cys sequence in a strand of sheet forms two disulfide bonds to a Cys-X-X-X-Cys sequence in a helix. Three-dimensional structures were generated using the torsion angle space program PEGASUS. The best ten structures had an average rmsd over all pairwise comparisons of 1.49 A. The average rmsd to a calculated average structure is 1.0 A. The resulting structures appear very similar to those of charybdotoxin, a related scorpion toxin.     

Hydrogen-deuterium substitution and solvent effects on the nitrogen-15 nuclear magnetic resonance of gramicidin S: evaluation of secondary structure.

Complete assignments of nitrogen-15 resonances of gramicidin S have been made in dimethyl sulfoxide, trifluoroethanol, and in a solvent mixture of dimethyl sulfoxide (50%) and methanol (50%). The assignments are achieved by utilizing the secondary structure of gramicidin S, by comparing the nitrogen-15 spectrum of gramicidin S with that of di-N-methylphenylalanine-gramicidin S and by taking into account the distinguishable value of nitrogen-15 chemical shift for valine in model compounds. Deuterium substitution for labile peptide protons was performed to delineate solvent shielded and deshielded peptide nitrogens and to substantiate further the signal assignments. The solvent titration on going from dimethyl sulfoxide to trifluoroethanol was also performed and shown to have a large deshielding effect on the peptide nitrogen whose corresponding peptide carbonyl, within the peptide moiety, was accessible to the trifluroethanol solvent.