3D-Structure of the interior fusion peptide of HGV/GBV-C by 1H NMR, CD and molecular dynamics studies.

In this work, we present a structural characterization of the putative fusion peptide E2(279-298) corresponding to the E2 envelope protein of the HGV/GBV-C virus by (1)H NMR, CD and MD studies performed in H(2)O/TFE and in lipid model membranes. The peptide is largely unstructured in water, whereas in H(2)O/TFE and in model membranes it adopts an helical structure (approximately 65-70%). The partitioning free energy DeltaG ranges from -6 to -7.5 kcal mol(-1). OCD measurements on peptide-containing hydrated and oriented lipid multilayers showed that the peptide adopts a predominantly surface orientation. The (1)H NMR data (observed NOEs, deuterium exchange rates, Halpha chemical shift index and vicinal coupling constants) and the molecular dynamics calculations support the conclusions that the peptide adopts a stable helix in the C-terminal 9-18 residues slightly inserted into the lipid bilayer and a major mobility in the amino terminus of the sequence (1-8 residues).

Structural characterization of a dipeptide compound with immunostimulant activity: 3-(5-thioxo-L-prolyl)-L-thiazolidine-4-carboxylic acid.

The structural characteristics of an immunostimulating agent (3-(5-thioxo-L-prolyl)-L-thiazolidine-4-carboxylic acid) have been established using a combination of 1H and 13C NMR spectroscopy, molecular mechanic calculations (in vacuo and in solution) and X-ray crystallographic analyses. Conformational calculations and NMR spectra identify two classes of conformers, cis and trans, around the peptide bond between the rings, while in the solid state only the cis form has been found.

Phenyl sulfur mustard derivatives of distamycin A.

The design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A are described and structure activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corresponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cinnamoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more potent than melphalan.

17beta-O-Aminoalkyloximes of 5beta-androstane-3beta,14beta-diol with digitalis-like activity: synthesis, cardiotonic activity, structure-activity relationships, and molecular modeling of the Na(+),K(+)-ATPase receptor.

A series of digitalis-like compounds with a 17-aminoalkoxyiminoalkyl or -alkenyl substituent was synthesized and evaluated for inhibition of Na(+),K(+)-ATPase and for inotropic activity. The highest inhibition was found with compounds having the substituent in configuration 17beta and the amino group at a distance of 6 or 7 bonds from C(17) of the digitoxigenin skeleton. The presence of the oxime function strengthens the interaction with the receptor, more if alpha,beta-unsaturated, thus mimicking the electronic situation of the unsaturated lactone in natural digitalis compounds. The most active compounds showed Na(+),K(+)-ATPase inhibitory potencies (IC(50)) 17-25 times higher than the standards digitoxigenin and digoxin and 3-11 times higher inotropic potencies (EC(50)) in isolated guinea pig left atria. These features are supported by a molecular model suggesting the possible interactions of the groups described above with particular amino acid residues in the H1-H2 domains of Na(+),K(+)-ATPase. Some interactions are the classical ones already described in the literature; a new, very strong interaction of the basic group with the Cys138 was found and adds new possibilities to design compounds interacting with this region of the receptor. The most interesting compounds were also studied in vivo in the anesthetized guinea pig for evaluating their inotropic effect versus the lethal dose. Compounds 9 and 12 showed a slightly higher safety ratio than digoxin and deserve further evaluation.

Tetracycline affects abnormal properties of synthetic PrP peptides and PrP(Sc) in vitro.

Prion diseases are characterized by the accumulation of altered forms of the prion protein (termed PrP(Sc)) in the brain. Unlike the normal protein, PrP(Sc) isoforms have a high content of beta-sheet secondary structure, are protease-resistant, and form insoluble aggregates and amyloid fibrils. Evidence indicates that they are responsible for neuropathological changes (i.e. nerve cell degeneration and glial cell activation) and transmissibility of the disease process. Here, we show that the antibiotic tetracycline: (i) binds to amyloid fibrils generated by synthetic peptides corresponding to residues 106-126 and 82-146 of human PrP; (ii) hinders assembly of these peptides into amyloid fibrils; (iii) reverts the protease resistance of PrP peptide aggregates and PrP(Sc) extracted from brain tissue of patients with Creutzfeldt-Jakob disease; (iv) prevents neuronal death and astrocyte proliferation induced by PrP peptides in vitro. NMR spectroscopy revealed several through-space interactions between aromatic protons of tetracycline and side-chain protons of Ala(117-119), Val(121-122) and Leu(125) of PrP 106-126. These properties make tetracycline a prototype of compounds with the potential of inactivating the pathogenic forms of PrP.

Determination of solution conformations of PrP106-126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics.

Experimental two-dimensional 1H NMR data have been obtained for PrP106-128 under the following solvent conditions: deionized water/2, 2,2-trifluoroethanol 50 : 50 (v/v) and dimethylsulfoxide. These data were analyzed by restrained molecular mechanics calculations to determine how changes in solvation affect the conformation of the peptide. In deionized water at pH 3.5, the peptide adopted a helical conformation in the hydrophobic region spanning residues Met112-Leu125, with the most populated helical region corresponding to the Ala115-Ala119 segment ( approximately 10%). In trifluoroethanol/H2O, the alpha-helix increased in population especially in the Gly119-Val122 tract ( approximately 25%). The conformation of this region was found to be remarkably sensitive to pH, as the Ala120-Gly124 tract shifted to an extended conformation at pH 7. In dimethylsulfoxide, the hydrophobic cluster adopted a prevalently extended conformation. For all tested solvents the region spanning residues Asn108-Met112 was present in a 'turn-like' conformation and included His111, situated just before the starting point of the alpha-helix. Rather than by conformational changes, the effect of His111 is exerted by changes in its hydrophobicity, triggering aggregation. The amphiphilic properties and the pH-dependent ionizable side-chain of His111 may thus be important for the modulation of the conformational mobility and heterogeneity of PrP106-126.

Nuclear magnetic resonance structure of d(GCATATGATAG). d(CTATCATATGC): a consensus sequence for promoters recognized by sigma K RNA polymerase.

The three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), from the promoter region of a gene regulating sporulation in Bacillus subtilis mother cells, was determined utilizing two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (2QF-COSY) spectra. To minimize the effect of methods used to obtain restraints and refine structure, several variables were studied. Interproton distance bounds were calculated very conservatively by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable and for nonexchangeable protons at different mixing times, assuming different overall correlation times and different starting structures. The 435 distance restraints were used with two different structural refinement methods: restrained molecular dynamics (rMD) and restrained Monte Carlo calculations (rMC). Refinement using different procedures and starting structures resulted in essentially the same structure (<0.8 A rmsd), indicating that the structure is defined by experimental restraints and not the refinement method or variables used. R factors indicate the structures fit the experimental NOE data very well. Some helical parameters, notably large negative X displacement, are characteristic of A-DNA, but others are characteristic of B-DNA. As with TG.CA steps in other duplex DNA sequences studied in our laboratory, the two TG.CA steps have a positive roll, with T6-G7 exhibiting the largest, and consequently a bent helix axis. The converged structure represents a time-averaged structure. However, multiple conformations, especially in deoxyriboses, were evident from vicinal coupling constants obtained from quantitative simulations of 2QF-COSY cross-peaks and from persistent inconsistencies in experimental distances due to nonlinear conformational averaging.

Cluster-iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states.

Site-directed mutants of adrenodoxin were studied for their ability to undergo cluster-iron substitution when reacted with zinc or cadmium salts under non-denaturing conditions in the presence or absence of reductants. Equilibrium and kinetic data for metal substitution were correlated with data on the stability to thermal unfolding and with the redox potential of the protein. Similarly to the wild-type protein, all mutants were able to stabilize a substituted form of the protein containing two metal (Zn or Cd) atoms and two sulfide ions/mol protein and a substituted form of the protein containing two sulfide ions and five Cd atoms/mol protein. However, the distribution of these two metal-substituted forms was different among the investigated proteins. [Ser95]Adrenodoxin stabilized either metal-substituted forms, confirming that Cys95 is not involved in metal coordination, even when five Cd atoms are bound to the protein. Removal of the extremely conserved hydroxy function at position 54 resulted in complete apoprotein formation upon reaction with Cd (75 % with Zn) under reducing conditions, indicating a cluster-harboring role for this function, which is conserved in all known 2Fe-2S proteins. Mutants at His56, which represents a residue unique to most vertebrate-type ferredoxins, were much more reactive than the wild-type protein with either metal, indicating that His56 plays a prominent role in the stabilization of the protein structure in the immediate vicinity of the cluster in this class of proteins. The nature of the metal-substitution products was dependent on cluster accessibility. For the reduced proteins, apoprotein formation depended on protein stability, while the velocity of metal substitution depended on the ease of cluster reduction.

Reversible, non-denaturing metal substitution in bovine adrenodoxin and spinach ferredoxin and the different reactivities of [2Fe-2S]-cluster-containing proteins.

The non-denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed-metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+ and Cd2+ was found to react also with oxidized adrenodoxin. Metal-treated proteins were resolved into a mixture of apoprotein, metal-substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal-substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium-substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal-substituted proteins could be re-converted into the native iron-containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.

Geometrical and conformational properties of ganglioside GalNAc-GD1a, IV4GalNAcIV3Neu5AcII3Neu5AcGgOse4Cer.

The aggregative properties of GalNAc-GD1a ganglioside, in comparison with those of GD1a, have been investigated and correlated to the intrinsic conformation and mobility of the oligosaccharide chain of the molecules. Micellar parameters in aqueous solution (molecular mass, hydrodynamic radius as well as the surface area at the lipid/water interface and the packing parameter of the monomer inserted in the aggregate) are measured by the laser light-scattering technique. The presence of a further GalNAc residue causes a 22% increase in molecular mass, contrary to expectation. Oligosaccharide moiety three-dimensional structures have been modeled using molecular mechanics and dynamics calculations, based on NOE interactions observed for native gangliosides dissolved in deuterated dimethylsulfoxide or, as mixed micelles with fully deuterated dodecylphosphocholine, in D2O. Compared with GD1a the GalNAc-GD1a is less mobile, thus influencing the surface area, this lower mobility together with the GalNAc-GD1a conformation leads to a larger number of monomers participating in the formation of the micelle. The results further substantiate the model in which the three-dimensional structure and the intrinsic dynamic properties of the oligosaccharide chain affect the geometrical properties of the aggregate.

Three dimensional structure of GD1b and GD1b-monolactone gangliosides in dimethylsulphoxide: a nuclear Overhauser effect investigation supported by molecular dynamics calculations.

A comparative study on the conformational features of the oligosaccharide moiety of GD1b and GD1b lactone gangliosides, in dimethylsulphoxide, has been carried out by nuclear Overhauser effect investigation; the experimental interresidue contacts have been used for restrained molecular mechanics and dynamics calculations. For GD1b, the tetrasaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2 ----8)-alpha-Neu5Ac-(2----3)]-beta-Gal has a circular arrangement leaving a highly hydrophobic region with seven hydrogens pointing towards the center. At one side of this region the three electron rich groups GalNAc--NH, external Neu5Ac--OH4 and internal Neu5Ac--COO- are grouped together; at the other side five polar groups (four hydroxy groups and the external Neu5Ac carboxylate) define a large annular hydrophilic region. The external Neu5Ac is close to the external Gal residue, and the external Neu5Ac--COO- is within van der Waals contact with the inner Neu5Ac-OH9 group. The beta-Gal-(1----3)-beta-GalNAc glycosidic linkage shows a high degree of freedom. For GD1b-L, the trisaccharide beta-GalNAc-(1----4)-[alpha-Neu5Ac-(2----3)]-beta-Gal is disposed to forming rigid partially circular arrangement showing strong interresidue contacts between the inner Neu5Ac-H8 and both GalNAc-H1 and GalNAc-H5. The conformation of the lactone ring is the boat 9(A),2(B)B. The lactonization of the disialosyl residue induces a strong variation of the preexisting torsional glycosidic angles phi and psi, leaving the external Neu5Ac far from the external Gal. In both GD1b and GD1b lactone gangliosides, the conformation of the sialic acid side chain is the same as that of the free sialic acid in which the H7 is trans to H8 and gauche to H6, thus indicating that the presence of glycosidic and/or ester linkages does not affect the conformational properties of sialic acid. Both GD1b and GD1b lactone containing sialic acid carboxylate anion(s) or undissociated carboxyl group(s) show the same three dimensional structure, indicating that the presence of charges does not affect the intrinsic conformational features of gangliosides.

Response to Anoxia in Rice and Wheat Seedlings: Changes in the pH of Intracellular Compartments, Glucose-6-Phosphate Level, and Metabolic Rate.

(31)P nuclear magnetic resonance spectroscopy was used to measure intracellular pH in living tissues. Oxygen deprivation caused fast cytoplasmic acidification from pH 7.4 to 7.0 in shoots of rice, Oryza sativa L. var arborio, a species highly resistant to anoxia. Acidification was complete after 10 minutes of anoxia. Alkalinization of both cytosplasm and vacuole followed thereafter. In the anoxia intolerant wheat shoots, Triticum aestivum L. var MEK, the same treatment caused a sharper cytoplasmic acidification, from pH 7.4 to 6.6, which occurred during a period of 2 hours. Cytoplasmic acidification continued with progress of anoxia and there was no vacuolar alkalinization comparable to the one observed in rice. In wheat oxyen, withdrawal also caused the reduction of both glucose-6-phosphate level and of metabolic rate. It also induced heavy losses of inorganic phosphate from tissues. Conversely, in rice, glucose-6-phosphate level and metabolic rate were increased and inorganic phosphate leakage from tissues was completely absent. These results are discussed in relation to the mechanisms of plant resistance to anoxia.

1H-NMR study and structure determination of 4,4- and 4,6-dimers from electrochemical reduction of NADP+.

The products arising from one-electron electrochemical reduction of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) have been studied by HPLC chromatography and 1H-NMR spectroscopy. HPLC and NMR analyses have shown seven dimeric species, the most abundant of which (40%) has been isolated and has resulted to be an NADP 4,4-linked dimer. The other two diastereoisomeric 4,4-dimers present for the 25% and 10%, respectively, have been detected in the crude reaction mixture, but have not been isolated. The 4,4-tetrahydrobipyridine structure and the stereochemistry at the ring-ring junction for these three isomers have been determined on the basis of their NMR parameters. Preparative HPLC chromatography also led to two fractions enriched in another four dimers, present in the crude mixture, which turned out to have a 4,6-tetrahydrobipyridine structure. All the chemical shifts and the H,H coupling constants of the 4,4- and 4,6-tetrahydrobipyridine systems have been obtained for the seven compounds. For the most abundant among the 4,4-dimers the NMR analysis also gave the coupling constant values of the ribose-diphosphate chain.

1H-, 13C-, 31P-NMR studies and conformational analysis of NADP+, NADPH coenzymes and of dimers from electrochemical reduction of NADP+.

All H,H, H,P and several C,P coupling constants, including those between C-4' and the vicinal phosphorus atom, have been determined for NADP+, NADPH coenzymes and for a 4,4-dimer obtained from one-electron electrochemical reduction of NADP+. From these data the preferred conformation of the ribose, that of the 1,4-dihydronicotinamide rings, and the conformation about bonds C(4')-C(5') and C(5')-O(5') were deduced. The preferred form of the 1,4- and 1,6-dihydropyridine rings and the conformation about the ring-ring junction were also obtained for all the other 4,4- and 4,6-dimers formed in the same reduction. All the dimers show a puckered structure, i.e., a boat form for the 1,4- and a twist-boat for the 1,6-dihydronicotinamide ring; both protons at the ring-ring junctions are equatorial and have preferred gauche orientation. On the contrary, the reduced coenzyme NADPH displays a planar or highly flexible conformation, rapidly flipping between two limiting boat structures. The conformation of the ribose rings, already suggested for the NADP coenzymes to be an equilibrium mixture of C(2')-endo (S-type) and C(3')-endo (N-type) puckering modes, has been reexamined by using the Altona procedure and the relative proportion of the two modes has been obtained. The S and N families of conformers have almost equal population for the adenine-ribose, whereas for the nicotinamide-ribose rings the S-type reaches the 90%. The rotation about the ester bond C(5')-O(5') and about C(4')-C(5'), defined by torsion angles beta and gamma respectively, displays a constant high preference for the trans conformer beta t (75-80%), whereas the rotamers gamma are spread out in a range of different populations. The values are distributed between the gauche gamma + (48-69%) and the trans gamma t forms (28-73%). The gamma + conformer reaches a 90% value in the case of NADP+ and NMN+. The conformations of the mononucleotides 5'-AMP, NMN+ and NMNH were also calculated from the experimental coupling constant values of the literature.

31P NMR study of daunorubicin-d(CGTACG) complex in solution. Evidence of the intercalation sites.

The interaction of daunorubicin with the self-complementary DNA fragment d(CGTACG) was studied by 31P NMR spectroscopy. The individual phosphates have been assigned for the nucleotide and the complex and signals from bound and free species in slow exchange at 19 degrees C were detected. In solution, the hexanucleotide binds two molecules of daunorubicin, which intercalate in the d(CG) sequence at both ends of the helix. Evidence for local deformations of the backbone at the sites of C5pG6, C1pG2 and G2pT3 phosphates is given. The binding constants for the stepwise equilibrium and the rate of dissociation of the intercalated duplex were also determined.

Metal ion binding to parvalbumin. A proton NMR study.

The 1H NMR spectra of carp parvalbumin saturated with Ca2+, Cd2+, La3+ and Lu3+ were compared, using 2D 1H NMR techniques as well as conventional 1H NMR spectra. The Ca2+ and Cd2+ saturated parvalbumin (with both high affinity Ca2+-binding sites occupied) gave rise to very similar spectra. This shows that these two species have almost identical protein conformations. The 1H NMR spectrum from the Ln3+ saturated parvalbumins deviated from the other two and it was therefore concluded that Cd2+ is a better probe for Ca2+ than Ln3+ in parvalbumin and probably also for related calcium binding proteins. The addition of excess of divalent metal ions, such as Mg2+ or Ca2+, causes small changes in the chemical shift of some methyl resonances. This is presumably caused by binding of these metal ions to a third site close to the CD site which is made up of the carboxylic groups from Glu 60 and Asp 61.

Kinetics of electron transfer between mitochondrial cytochrome c and iron hexacyanides.

The reduction of horse and Candida krusei cytochromes c by ferrocyanide has been studied by 1H NMR spectroscopy and the reaction found to involve a precursor complex of ferrocyanide bound to ferricytochrome c (pH* 7.4, 2H2O, I = 0.12, and 25 degrees C). The electron transfer rate constants for the reduction of the two ferricytochromes by associated ferrocyanide were found to be the same at 780 +/- 80 sec-1 but the association constants for binding of ferrocyanide to ferricytochrome c were significantly different: horse, 90 +/- 20 M-1 and Candida, 285 +/- 30 M-1. The different association constants partly accounts for the previously observed reactivity difference between horse and Candida cytochromes c. Comparison of the NMR data with data obtained by other kinetic methods has allowed the electron transfer rate constant for the oxidation of ferrocytochrome c by associated ferricyanide to be determined. This was found to be 4.6 +/- 1 X 10(4) sec-1.

Association constants for metal hexacyanide binding to cytochrome c.

The binding of [Co(CN)6]3-, and that of [Fe(CN)6]3- and [Ru(CN)6]4- using a competitive method, to horse cytochrome c has been studied by 59Co NMR spectroscopy. At I = 0.07 M, without added salt and in 2H2O at pH* 7.3 (measured in 2H2O) and 25 degrees C, there are at least two binding sites on ferricytochrome c and ferrocytochrome c for [Co(CN)6]3-. Association constants were determined to be 2.0 +/- 0.6 X 10(3) M-1 and 1.5 +/- 0.5 X 10(2) M-1, respectively, with no effect of the oxidation state of the cytochrome. At higher ionic strength (I = 0.12 M) adjusted with KCl the binding markedly decreased, and, although it was not possible to determine the precise binding stoichiometry and magnitude of association constants, it is clear that the association constants are less than or equal to 1.5 X 10(2) M-1. The binding of [Ru(CN)6]4- at I = 0.07, without added salt and in 2H2O at pH* 7.3 and 23 degrees C, was not precisely defined, but its binding strength relative to that of [Fe(CN)6]3- was determined. Extrapolating this to I = 0.12 (KCl) suggests that under these conditions the association constant for [Ru(CN)6]4- binding to ferricytochrome c is less than or equal to 3 X 10(2) M-1.