Biological and conformational studies on analogues of a synthetic peptide enhancing HIV-1 infection

We have previously demonstrated that a 23-amino acid peptide derived from the V3 loop of the surface glycoprotein of the HIV-1 strain MN is able to bind CD4 and to enhance HIV-1 infection. Further studies have suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. This paper describes the biological and physico-chemical characterization of three analogues of reduced sequence that have been designed in order to identify the minimum active sequence of this peptide corresponding to the MN-HIV-1 principal neutralizing domain. Biological studies indicate that the entire sequence is required for biological activity and that the sequence 1–18 presents an inhibitory activity. CD and FT-IR absorption data are discussed here in order to identify possible structure-function correlations. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

1H-nuclear magnetic resonance studies of the neuropeptide head activator

The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, kex, of the backbone amide protons and the correspondent activation enthalpies, delta H not equal to, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s-1 at 274 K, the activation enthalpies from 60 to 75 kJ.mol-1. The pK values of the terminal carboxyl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel beta-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

Hydrogen-1 and carbon-13 nuclear magnetic resonance conformational studies of the His-Pro peptide bond: conformational behavior of TRH

Cis/trans isomerism of the His-Pro peptide bond provides a convenient model for the effect of a slow conformational change which may have wider biological significance. Above the imidazole pK, His-Pro is conformationally analogous to the (isosteric) peptide Phe-Pro. Protonation of the imidazole sidechain is associated with a large decrease in the cis/trans ratio. Detailed 1H and 13C n.m.r. analysis suggests the importance of electrostatic/hydrogen bonding interactions between the charged imidazolium sidechain and the proline carboxyl as the basis for this effect. In contrast to a previous report, cis/trans isomerism in TRH is shown to be related to titration of the imidazole sidechain, exhibiting a pK of 6.1.     

13C- and 1H-nuclear magnetic resonance spectroscopy of permethylated disaccharides

The equilibrium composition of D-psicose in water, as determined from its ¹³C n.m.r. spectrum, is 7:2:5:5 α-furanose:β-furanose:α-pyranose:β-pyranose. These data, which are discussed in relation to the anomeric and ring-form equilibria of the other 2-hexuloses, are in general agreement with expectations based on conformational analysis. However, although the ¹³C chemical-shift pattern of the β-pyranose is closely consistent with the 1C(D) conformation predicted for this anomer, that of the α-pyranose is less readily reconciled with its predicted C1(D) conformation. Usually, carbon-13 nuclei of the furanose rings are substantially less shielded than those of their pyranose counterparts; for 2-hexulopyranoses in general, overall ¹³C shielding is close to that of those aldopyranoses expected to have similar conformational free-energies. Spectral data are also reported for several derivatives (glycosides, ethers, and selectively deuterated compounds) that were utilized in analysis of the D-psicose spectrum.     

1H-nuclear magnetic resonance spectroscopy of reducing-residue anomeric protons of pertrifluoroacetylated carbohydrates

Eight monosaccharides (L-arabinoside, L-fucose, D-galactose, D-glucose, D-lyxose, D-mannose, L-rhamnose, and D-xylose), eight disaccharides (cellobiose, gentiobiose, isomaltose, lactose, maltose, nigerose, sophorose, and xylobiose), and three trisaccharides (isomaltotriose, maltotriose, and xylotriose) were derivatized with N-methylbis-(trifluoroacetamide) in pyridine solution to form trifluoroacetylated derivatives. These were analyzed by 1H-n.m.r. spectroscopy to determine the characteristics of the spectra and distributions of the reaction products. Peaks corresponding to reducing-residue anomeric protons were located significantly downfield of all others, and were in general 0.4 p.p.m. or more downfield of equivalent signals from the same carbohydrates when they were free or derivatized with other groups. Neither the location of anomeric proton peaks relative to each other nor the degree of spin-spin coupling between H-1 and H-2 varied greatly with type of derivatization. Spin-spin coupling, however, decreased for some beta-pyranose forms of xylobiose and the three trisaccharides. In all examples except some where H-2 was oriented equatorially to a pyranose ring, the proportion of the alpha-pyranose was either enhanced or not changed in concentration by trifluoroacetylation.     

Multinuclear magnetic resonance studies on serine protease transition state analogues

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.     

1H-nuclear magnetic resonance of intercalators and rGCA: a potential mutagenicity probe

Variable temperature 1H-nuclear magnetic resonance (NMR) has been used to study the interaction of the RNA trimer, GpCpA, with the intercalators ethidium bromide and the acridine derivatives; proflavin, 9-amino-acridine, acridine orange, acridine yellow and acriflavin. The complexes formed were studied at nucleic acid to drug ratios of 1:1 and 5:1, the latter being useful in defining the effects of structural variation in the acridine series and in determining the site of intercalation. All the intercalators greatly stabilized the oligonucleotide duplex, the average melting temperature (Tm) increasing by up to 30 degrees C. Significant changes in individual Tms and chemical shifts were observed for all the GpCpA protons. 9-Amino-acridine and acriflavin did not stabilize the GpCpA duplex as substantially as the other acridine derivatives. It is suggested that this intercalator:GpCpA system, and its associated NMR-derived Tm, is a useful physical probe for potential mutagens.     

Characterisation of human embryonic stem cells conditioning media by 1H-nuclear magnetic resonance spectroscopy

Background

Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking.

Methodology/Principal Findings

Using a ¹H-Nuclear Magnetic Resonance (¹H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the ¹H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at −20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine.

Conclusions/Significance

¹H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.     

The structural isomerisation of human-muscle adenylate kinase as studied by 1H-nuclear magnetic resonance

Human muscle adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3.) was studied by 1H-nuclear magnetic resonance spectroscopy. The C-2 and C-4 proton resonances of the active-center histidine His-36 could be identified; the pK of His-36 was determined as 6.1. The pK of His-189 is very low (4.9) although it is located at the surface of the protein. Other resonance lines are discussed in comparison with NMR spectra of porcine adenylate kinase [McDonald et al. (1975) J. Biol. Chem. 250, 6947-6954]. A pH-dependent structural isomerization as shown by X-ray crystallography in the pig enzyme [Pai et al. (1977) J. Mol. Biol. 114, 37-45] was not observed for human adenylate kinase in solution. However, the binding of adenosine(5')pentaphospho(5')adenosine (Ap5A), a bisubstrate inhibitor, to adenylate kinase causes an overall change of the NMR spectrum indicative of a large conformational change of the enzyme. The exchange rate (koff) for Ap5A was estimated as 10 s-1 and decreases by addition of Mg2+. On the basis of these values and the known dissociation constant it is likely that the binding of Ap5A is a diffusion-controlled process kon being 10(8) M-1 s-1. In conclusion, the system Ap5A/Mg2+/human adenylate kinase, which has been studied by NMR spectroscopy and X-ray diffraction in parallel, is suitable for analyzing the induced fit postulated by Jencks for all kinase-catalyzed reactions.     

Proton magnetic resonance studies on dermorphin and its peptide fragments

Dermorphin (Tyr? D-Ala? Phe? Gly? Tyr? Pro? Ser? NH₂), a potent natural peptide opioid, its synthetic L-Ala² analog, and all the N fragments from the tripeptide (Tyr? D -Ala? Phe? NH₂) to the parent hexapeptide amide were characterized for the first time by means of proton nmr spectroscopy at 11.74 T. Assignments of most protons of dermorphin were facilitated by the study of the N-terminal fragments. Comparison of spectroscopic parameters with relative pharmacological activity is proposed as a possible means of studying flexible agonists in solution.     

X-ray conformational study of hydrazino peptide analogues

We have solved the crystal structures of nine pseudo-peptide analogues deriving from the hydrazino analogue of glycine or valine (N beta H2-N alpha H-C alpha HR-CO2H, R = H or iPr) or proline (N beta H2-N alpha-C alpha H-CO2H) and containing the hydrazide (CO-N beta H-N alpha less than) or N beta-Z-aminoamide [formula; see text] [CO-N alpha(N beta HZ)] peptidomimetic link. This study gives access to the average geometry of these two links, to their inter- and intramolecular interaction modes, and to their influence on the conformational properties of the molecules.     

Adenine nucleotide N-glycosidase activity of the A-chain of cinnamomin characterized by 1H-nuclear magnetic resonance

Plant ribosome-inactivating proteins specifically cleave an N-glycosidic bond of a unique adenosine in the largest ribosomal RNA, releasing an adenine from ribosomes of different sources. Here, 1H-nuclear magnetic resonance is used to analyze the enzymatic products of the A-chain of cinnamomin, a type-II ribosome-inactivating protein (RIP) acting on the nucleotides in situ. The enzymatic activities of the RIP on nine nucleotides are compared. Cinnamomin A-chain can cleave the N-glycosidic bond and release an adenine base from adenine nucleotides except 5'-ATP; however, it cannot act on 5'-GMP, 5'-CMP, and 5'-UMP. The A-chain in the mixture of cinnamomin A- and B-chain exhibits higher activity toward adenine nucleotides than the A-chain alone does, suggesting that the B-chain can conformationally stabilize the A-chain. Intact cinnamomin also exhibits lower activity toward adenine nucleotides. However, cinnamomin B-chain and heat-denatured intact cinnamomin cannot hydrolyze all the tested nucleotides. We conclude that hydrolysis of the N-C glycosidic bond of nucleotide compounds by cinnamomin A-chain has a base preference, and the negatively charged phosphate group(s) reduces the recognition ability of the A-chain to adenine nucleotide.     

Proton magnetic resonance studies of the conformational changes of dideoxynucleoside ethyl phosphotriesters

PMR investigations on the diastereomeric phosphate methyl protons of the dinucleoside ethyl phosphotriesters Tp(C₂H₅)T, dA, and dIp(C₂H₅)dI have been used to study the conformational changes of these dimersin solution. In D₂O (273°K), the diastereomeric phosphate-methly groups of Tp(C₂H₅)T appear as a triplet. The methyl resonances of dIp(C₂H₅)dI and dAp(C₂H₅)dA appear as two sets of triplets and their chemical shift differences (δ₁ ? δ₂), decrease with increasing temperature, finally becoming zero at 292°K and 333°K, respectively. The same phenomenon is observed for dAp(C₂H₅)dA in CD₃OD; in this detacking solvent, the difference (δ₁ ? δ₂) diminishes to zero at a lower temperature (261°K). At room temperature in D₂O, the chemical shift of the phosphate methyl of Tp(C₂H₅)T appears at lower field than those of dIp(C₂H₅)dI or dAp(C₂H₅)dA. The differences between the chemical shifts of these groups (δ_I ? δ_T or δ_A ? δ_T) increase with increasing temperature, and reach maximal values at 301°K and 333°K, respectively. The results suggest that at low temperature the largest fraction of the dimer population exists in a stacked state, with the phosphate-ethyl groups outside the stack. Increasing temperature causes an oscillation of the bases and a shift in the dimer population away from the stacked state. Finally at high temperature, the planar bases rorate with respect to one another and in the case of dIp(C₂H₅)dI and dAp(C₂H₅)dA, the ethyl groups experience shielding by the anisotropic ring current of the five-membered ring of the bases. Thus, the current pmr studies and those reported earlier from our laboratory support an “oscillation-rotation model” for the unstacking process of the dimers. The relationship of this model and the “two-state model” is discussed.     

On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies

The relative importance of tyrosine and histidine residues for the catalytic action of Escherichia coli asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) was studied by chemical modification and 1H-NMR spectroscopy. We show that, under appropriate reaction conditions, N-bromosuccinimide (NBS) as well as diazonium-1H-tetrazole (DHT) inactivate by selectively modifying two tyrosine residues per asparaginase subunit without affecting histidyl moieties. We further show that diethyl pyrocarbonate (DEP), a reagent considered specific for histidine, also modifies tyrosine residues in asparaginase. Thus, inactivation of the enzyme by DEP is not indicative of histidine residues being involved in catalysis. In 1H-nuclear magnetic resonance (NMR) spectra of asparaginase signals from all three histidine residues were identified. By measuring the pH dependencies of these resonances, pKa values of 7.0 and 5.8 were derived for two of the histidines. Titration with aspartate which tightly binds to the enzyme at low pH strongly reduced the signal amplitude of the pKa 7 histidyl moiety as well as those of resonances of one or more tyrosine residues. This suggests that tyrosine and histidine are indeed constituents of the active site.     

NMR conformational studies on a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor

The combined use of several nuclear magnetic resonance and restrained molecular dynamics techniques allowed the formulation of a molecular model for the preferred solution conformation of a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor. In the model, the conformation of the 20-membered tocin ring, with the two Cys¹ and Cys⁶ residues bridged by a disulphide bond, is very close to that observed for isolated ocytocin in the solid state; in addition, a type II β-turn is postulated for the 7-10 segment of the acyclic tail containing the Lys¹¹-Arg¹² processing site, and connecting ocytocin to the neurophysin domain, while the C-terminal 13-20 segment of the molecule is believed to assume a helical structure. This particular structural organization could be important in participating as the favorable conformation for optimal substrate-enzyme active site recognition and processing by specific endoproteases. © 1996 John Wiley & Sons, Inc.     

1H-nuclear magnetic resonance (NMR) spectroscopic study of B lymphocytes stimulated with lipopolysaccharide (LPS)

1H-nuclear magnetic resonance (NMR) spectroscopy was applied to the study of glucose metabolism of B lymphocytes (B cells) activated with lipopolysaccharide (LPS) in a complex medium. The glucose in the medium is degraded to produce lactic acid by B cells activated with LPS to a far larger extent than by non-activated cells.