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The solution structure of a custom lytic peptide, cecropin B3 (CB3), having two identical hydrophobic segments on both the N- and C-termini, was investigated by two-dimensional NMR spectroscopy. The need to determine the structure of this peptide is rooted in its specific ability to lyse lipid layers that have a high content of anionic lipid. The lytic activities of CB3 on cell membranes including cancer cells and bacteria is found to be less than cecropin B1. The results show that CB3 has four discrete segments forming α helical structures. The crumpled structure of CB3 provides evidence for the lysis of the lipid layer being via a pathway that differs from pore formation. The results in this study provide strong clues towards a rational design for a potent antimicrobial and antitumor peptide. [ABSTRACT FROM AUTHOR]

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences. Both precursor proteins include a cleavable N-terminal leader of 25 amino acids. N-terminal sequencing of PAO purified from barley seedlings reveals a unique amino-acid sequence corresponding to the BPAO2 N-terminus as predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO-specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C-terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles. [ABSTRACT FROM AUTHOR]

A model based on the Poisson–Boltzmann equation has been used to model electrostatics in Anti-p24 (HIV-1) Fab–antigen association. The ionization state at different pH values has been simulated and the results have been used to estimate the stability at different pH values and to generate electrostatic potential maps at physiological ionic strength. The analysis of the electrostatic potential at the solvent-accessible surface shows that residues involved in binding are mostly found in the highest, but also in lowest potential regions. Brownian dynamics simulations have been used to estimate the enhancement of the association rate due to electrostatics which appears limited (≈ 2 at 150 mm ionic strength and ≈ 3 at 15 mm ionic strength). A much more pronounced effect is observed upon increase of the charge of the diffusing particle. These results compare well with results obtained previously in similar studies on different systems and may serve to estimate the expected order of magnitude of electrostatic effects on association rates in antibody–antigen systems. [ABSTRACT FROM AUTHOR]

The microcystin-RR structures are compared with the structures of microcystin-LR in solution as well as in the crystal structure of the complex with protein phosphatase. The gross structures of the two peptides are similar, but with a more accentuated and compact saddle structure for microcystin-RR. The structural differences affect the hydrogen-bond pattern in the peptides and the location of the side chain of N-methyldehydroalanine, both of which are important for the ability of the peptide to form a tight complex with protein phosphatase. These structural differences may contribute to the observed differences in toxicity of microcystin-RR and microcystin-LR. [ABSTRACT FROM AUTHOR]

Reports on the solution structure of two mismatches in the K-ras gene context by nuclear magnetic resonance and molecular dynamics. Study of nuclear magnetic resonance spectra and molecular dynamics; Presence of duplexes in the gene coding sequence; Introduction of mismatches and their global confirmation.

Na+/K+-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylearbonyl-e-aminocaproate (HDMA), 3-amlno-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-O-succinyl-[2-(N-maleimido)]cthylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP, At 37°C, half-maximal inhibition of Na+/K+-ATPase was seen by HDMA at 0.47 μM, by ADHS at 5.8 μM, by lAD at 8 μM and by DSME at 94 μM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K+-ATPase. Affinity labeling of the a subunit by ‘front door’ or ‘back door’ phosphorylation was only seen with HDMA or ADHS in the range 0.1 μM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain. Tryptic hydrolysis of the HDMA-modified Na+/K+-AtPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cysl04 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J.-D., Louvard, D. & Rossier, B. C. (1992) EMBO J. II, 1681–1687], that a cysteine residue (probably Cysl04) participates in the structure and function of the cardiac glycoside binding. [ABSTRACT FROM AUTHOR]

A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca2+-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2′, S3, S5 and S6 subsites may also contribute to substrate binding. Based on this method, protein engineering can be employed not only to test the predicted enzyme-susbtrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors. [ABSTRACT FROM AUTHOR]

The solution structure of the human plasminogen kringle I domain complexed to the antifibrinolytic drug 6-aminohexanoic acid (∊Ahx) was obtained on the basis of [SUP1]H-NMR spectroscopic data and dynamical simulated annealing calculations. Two sets of structures were derived starting from (a) random coil conformations and (b) the (mutated) crystallographic structure of the homologous prothrombin kringle 1. The two sets display essentially the same backbone folding (pairwise root-mean-square deviation, 0.15 nm) indicating that, regardless of the initial structure, the data is sufficient to locate a conformation corresponding to an essentially unique energy minimum. The conformations of residues connected to prolines were localized to energetically preferred regions of the Ramachandran map. The Pro30 peptide bond is proposed to be cis. The ligand-binding site of the kringle I is a shallow cavity composed of Pro33, Phe36, Trp62, Tyrô4, Tyr72 and Tyr74. Doubly charged anionic and cationic centers configured by the side chains of Asp55 and Asp57, and Arg34 and Arg7l, respectively, contribute to anchoring the zwitterionic ∊Ahx molecule at the binding site. The ligand exhibits closer contacts with the kringle anionic centers (≈0.35 nm average O ... H distance between the Asp5S/Asp57 carboxylate and ligand amino groups) than with the cationic ones (≈0.52 nm closest O ... H distances between the ligand carboxylate and the Arg34/Arg7 l guanidino groups). The ∊Ahx hydrocarbon chain rests flanked by Pro33, Tyr64, Tyr72 and Tyr74 on one side and Phe36 on the other. Dipolar (Overhauser) connectivities indicate that the ligand aliphatic moiety establishes close contacts with the Phe36 and Trp62 aromatic rings. The computed structure suggests that the ∊Ahx molecule adopts a kinked conformation when complexed to kringle 1, effectively shortening its dipole length to ≈O.65 nm. [ABSTRACT FROM AUTHOR]

A one- and two-dimensional NMR study has been performed on seven A(2'-5')A(2'-5')A fragments containing 9-(3'-fluoro-3'-deoxy-β-D-xylofuranosyl)-adenine (AP) or 3'-fluoro-3'-deoxyadenosine (AP) residues at different positions, and on the corresponding monomers. A(2'-5')A(2'-5')A' served as a reference compound. The fluoro substituent governs the conformation of the sugar ring: an AP residue displays mainly N-type sugar and the ring is considerably flattened (θN ≈ 30°) comparedd to AP. residues (θ ≈ 40°), which exhibit almost pure S-type conformation. Moreover. in AP moieties the rotamer distribution around torsion angle γ-(05'-C5'-C4'-C3') and the base orientation are influenced to a large extent by the presence of the fluorine substituent. The sugar rings of nonflourinated residues in the trimers appear rather flexible. A possible correlation between the conformational characteristics of the fluorinated fragments and their biological activity has been found: the fragments that meet the prerequisites for binding to RNase L indeed show enhanced binding to this endonuclease. Furthermore, substitution of the 3'-OH group of the second residue by hydrogen or of the 3'-OH group of the 2'-terminal residue by fluorine or hydrogen results in increased resistance towards 2'-5'-phosphodiesterase. [ABSTRACT FROM AUTHOR]

We have studied the structures of synthetic peptides which correspond to the proposed first and second membrane-spanning segments of the human red cell anion transporter (band 3). The peptides, which were acetylated at their N-termini and amidated at the C-termini, comprise the 20 amino acids of residues 405-424 and 21 amino acids of residues 436-456 of the human band 3 sequence. The solution structures of the peptides in trifluoroethanol were studied by two-dimensional NMR spectroscopy. Characteristic NOEs were observed indicating that the peptides adopted a predominantly α-helical structure in trifluoroethanol solution. Dynamical simulated annealing using the program XPLOR was employed for the structure calculations. The amide exchange rates in trifluoroethanol have also been measured and are consistent with an α-helical structure for the peptides. [ABSTRACT FROM AUTHOR]

The solution conformation of sarcotoxin IA, which is an antibacterial peptide isolated from Sarcophaga peregrina with a molecular mass of 4 kDa, was determined by NMR spectroscopy and hybrid distance geometry/dynamical simulated annealing calculations. On the basis of 227 experimental constraints, including 185 distance constraints obtained from NOE and 42 constraints associated with 21 hydrogen bonds, a total of 18 converged structures of sarcotoxin IA were obtained. The final 18 converged structures exhibit backbone-atomic root-mean-square differences about the averaged coordinate positions of 0.070 +/- 0.027 nm for residues 3-23 and 0.040 +/- 0.017 nm for residues 28-38. It has been indicated that sarcotoxin IA consists of two amphiphilic alpha-helical regions, i.e. helix I (Leu3-Gln23) and helix II (Ala28-Ala38), with a hinge region (Gly24-Ile27), which connects helix I and helix II. We conclude that these two amphiphilic helical segments of sarcotoxin [ABSTRACT FROM AUTHOR]

Presents the complete main chain and approximately 75 per cent of the side chain H-NMR assignments of the 129-residue protein, turkey egg-white lysozyme. Hydrogen-exchange rates, chemical shifts and coupling constants; Comparison of the NH-alphaCH coupling constants of turkey lysozyme to torsion-angle data from three crystal structures of the protein.

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of CysT2 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cysl21 bonded to Cysl41 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed. [ABSTRACT FROM AUTHOR]

A conformational study of the double-stranded decanucleotide d(GCCG*G*ATCGC) · d(GCGATCCGGC), with the G* guanines chelating a cis-Pt(NH3)2 moiety, has been accomplished using ¹H and 31P NMR, and molecular mechanics. Correlation of the NMR data with molecular models has disclosed an equilibrium between several kinked conformations and has ruled out an unkinked structure. The deformation is localized at the CG*G* · CCG trinucleotide where the helix is kinked by approximately 6o° towards the major groove and unwound by 12 - 19°. The models revealed an unexpected mobility of the cytosine complementary to the 5'-G*. This cytosine can stack on either branch of the kinked complementary strand. The energy barrier between the two positions has been calculated to be ≤ 12 kJ/mol. The NMR data are in support of rapid flip-flopping of this cytosine. An explanation for the strong downfield shift observed in the 31P resonance of the G*pG* phosphate is given. [ABSTRACT FROM AUTHOR]

Two-dimensional NMR methods have been used to assign aromatic and methyl group resonances in the ¹H-NMR spectrum of oxidized uteroglobin. Assignments to specific amino acids are based on X-ray-determined structures of two crystal forms (C2221 and P21) and on an energy-minimized X-ray structure of the C2221 form of uteroglobin. These preliminary assignments are sufficient to probe the interaction of oxidized uteroglobin with progesterone in solution. The protein global structure is unmodified but some direct or indirect conformational changes are induced in the H1H4(H1′H4′) pockets and close to Phe28 by progesterone. [ABSTRACT FROM AUTHOR]

Both cis-[Pt(NH3)2(4-Me-Py)Cl]+ and trans-[Pt(NH3)2(4-Me-Py)Cl]+ bind DNA covalently at the N7 site of guanine residues forming mono-dentate adducts. However, like cisplatin and transplatin, only the cis isomer has anti-cancer activity, whereas the trans-isomer does not. In order to understand the molecular basis of the different activities associated with cis-[Pt(NH3)2(4-Me-Py)Cl]+ and trans-[Pt(NH3)2(4-Me-Py)Cl]+, the interactions of these two platinum compounds with the DNA heptamer CCTG*TCC :GGACAGG duplex (G* is the platinated guanine) have been examined. The reaction rate of cis-[Pt(NH3)2(4-Me-Py)Cl]+ with the single-stranded CCTGTCC is significantly faster than that of the trans isomer. The solution structure of the platinum-DNA adducts has been studied by two-dimensional NMR spectroscopy. Both the cis-platinum adducts and the trans-platinum adducts destabilize the DNA duplex significantly. The melting temperature (Tm) of the platinated heptamer duplex is estimated to be 10 +C lower than for the unplatinated duplex by NMR. At 2 +C, the base pairs located on the 5′ side of the oligonucleotide, beyond the platinum lesion site, are disrupted. Over time, the platinum-DNA complex decomposes and the cis-[Pt(NH3)2(4-Me-Py)] platinum complex is gradually detached from DNA. No interstrand crosslinking is observed. The biological implications of the structural studies are discussed. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Lys7p was proposed to be the enzyme catalyzing the dehydratation of homocitrate to cis-homoaconitate, the second step of the lysine biosynthetic pathway. In this communication we provide evidence that Lys7p is involved in oxidative stress protection. Cells deleted for the LYS7 gene displayed, in addition to lysine auxotrophy, methionine auxotrophy, sensitivity to superoxide generating drugs and light irradiation, and diminution of calcineurin activity. The SOD1 gene encoding the Cu/Zn-superoxide dismutase was expressed in strains lacking Lys7p, and although Sod1p was produced in normal amounts no detectable enzyme activity was found. In contrast, the mitochondrial Mn-superoxide dismutase activity did not seem to be impaired. lys7 cells exhibited a normal uptake of Cu from growth medium. The Cu/Zn-superoxide dismutase activity was restored by addition of Cu (but not by addition of other metallic cations) to the growth medium or to cellular extracts, suggesting a lack of Cu2+ at the active site. These results render it necessary to reconsider the role of the Lys7p. Its involvement in Cu metabolism and oxidative-stress protection, and the possibility of a human equivalent in amyotrophic lateral sclerosis are discussed. [ABSTRACT FROM AUTHOR]

Cytochrome P-450cam oxidises 1,2,3,4-tetrahydro-β-carboline, an indolic alkaloid. We report here measurements of the product distribution of this oxidation. To rationalise the experimental results, ab initio quantum-chemistry calculations of the product stabilities and molecular-dynamics calculations of the substrate-binding mode in the active site were performed. The calculations suggest that the product distribution is influenced by both the relative intrinsic gas-phase stabilities of the monohydroxy products and by conformational rearrangement of the active site on substrate binding. [ABSTRACT FROM AUTHOR]

Secretoneurin is a 33-amino-acid polypeptide generated by proteolytic cleavage of secretogranin II at paired dibasic sequences. It has recently been shown that secretoneurin exerts biological activities such as stimulation of dopamine release from striatal neurons and activation of monocyte migration, suggesting that the peptide may modulate both neurotransmission and inflammatory response. In the present study, we have investigated the conformation of synthetic secretoneurin in methanol solution by two-dimensional 1H-NMR, circular dichroism and molecular modeling. Using sequential information, specific assignments have been made for resonances arising from all protons, except for the labile proton of the N- terminal Thr of the peptide. The solution structure of secretoneurin has been deter mined by distance geometry and restrained molecular dynamics, using distance and dihedral constraints derived from the NMR data. The conformation obtained is composed of two contiguous a-helices comprising residues Glu3-Gln8 and Prol11 -Gly25. An excellent concordance was observed between these conformational data and prediction with the AGADIR program for the location for the helices in the sequence. These conformational data should help to elucidate the involvement of the tertiary interactions and to design secretoneurin analogs. [ABSTRACT FROM AUTHOR]

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D [sup 1]H NMR spectroscopy. TVIIA contains six cysteine residues which form a ‘four-loop’ structural framework common to many peptides from Conus venoms including the ω-, δ-, κ-, and µO-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2–27 of 0.18 ± 0.05 Å for the backbone atoms and 1.39 ± 0.33 Å for all heavy atoms. The structure consists of a triple-stranded, antiparallel β sheet with +2x, -1 topology (residues 7–9, 16–20 and 23–27) and several β turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors. [ABSTRACT FROM AUTHOR]