Binding of peptides in solution by the Escherichia coli chaperone PapD as revealed using an inhibition ELISA and NMR spectroscopy

PapD is the prototype member of a family of periplasmic chaperones which are required for assembly of virulence associated pili in pathogenic, gram-negative bacteria. In the present investigation, an ELISA has been developed for evaluation of compounds as inhibitors of PapD. Synthetic peptides, including an octamer, derived from the C-terminus of the pilus adhesin PapG were able to inhibit PapD in the ELISA. Evaluation of a panel of octapeptides in the ELISA, in combination with NMR studies, showed that the peptides were bound as extended beta-strands by PapD in aqueous solution. The PapD-peptide complex was stabilized by backbone to backbone hydrogen bonds and interactions involving three hydrophobic peptide side chains. This structural information, together with previous crystal structure data, provides a starting point in efforts to design and synthesize compounds which bind to chaperones and interfere with pilus assembly in pathogenic bacteria.     

Conformation of an analog of human follicular gonadotropin releasing peptide TF14 in solution by 2D-NMR

Since the discovery of hF-GRP, several analogs have been synthesized in order to see their effects on the gonadotropin releasing activity, either as agonists or antagonists to this peptide. TF14 is one of these analogs, whose 14th position in the primary sequence is Phe instead of Asn in hF-GRP, while its activity is doubled. 2D-NMR (TOCSY, ROESY) was used to determine the conformation of TF14 in solution. Compared with hF-GRP, the whole peptide is in a non-typical more extended conformation, which may give some clue to the relation between structure and function of these two peptides.     

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.     

Polarization-enhanced NMR spectroscopy of biomolecules in frozen solution

Large dynamic nuclear polarization signal enhancements (up to a factor of 100) were obtained in the solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectra of arginine and the protein T4 lysozyme in frozen glycerol-water solutions with the use of dynamic nuclear polarization. Polarization was transferred from the unpaired electrons of nitroxide free radicals to nuclear spins through microwave irradiation near the electron paramagnetic resonance frequency. This approach may be a generally applicable signal enhancement scheme for the high-resolution solid-state NMR spectroscopy of biomolecules.     

The localisations in liposomal membranes of the tetrahydrofuran ring moieties of the annonaceous acetogenins, annonacin and sylvaticin, as determined by 1H NMR spectroscopy

We studied 34 apparently healthy children and 2 propositi from kindreds with familial juvenile hyperuricaemic nephropathy (FJHN) - a disorder characterised by early onset, hyperuricaemia, gout, familial renal disease and a similarly low urate clearance relative to glomerular filtration rate (GFR) [fractional excretion of uric acid (FEur) 5.1+/-1.6%] in young men and women. In addition to the propositi, 17 asymptomatic children were hyperuricaemic -- mean plasma urate (368+/-30 micromol/l), twice that of controls (154+/-41 micromol/l). Eight of them had a normal GFR ( > 80 ml/min per 1.73 m2), and 11 renal dysfunction, which was severe in 5. The FEur in the 14 hyperuricaemic children with a GFR > 50 ml/min was 5.0+/-0.5% and in the 5 with a GFR < or =50 ml/min was still low (11.5+/-0.2%) compared with controls (18.4+/-5.1%). The 17 normouricaemic children (185+/-37 micromol/l) had a normal GFR (>80 ml/min) and FEur (14.0+/-5.3%). The results highlight the dominant inheritance, absence of the usual child/adult difference in FEur in FJHN and presence of hyperuricaemia without renal disease in 42% of affected children, but not vice versa. Since early allopurinol treatment may retard progression to end-stage renal failure, screening of all relatives in FJHN kindreds is essential.     

Conformation of phosphatidylserine in bilayers as studied by Fourier transform infrared spectroscopy

The 13C labeled lipid 1[1'-13C]DPPS-NH4+ and its metal salts were used to unambiguously assign all carbonyl vibrations in the infrared spectrum of phosphatidylserines. It is shown that the C=O stretching band at 1741 cm-1 of phosphatidylserines previously assigned to the sn-1 C = O vibration contains contributions from both the sn-1 and the sn-2 carbonyls. The C=O stretching band at frequencies between 1715 and 1730 cm-1 previously assigned to the sn-2 C=O vibration also contains contributions from both carbonyl groups. The frequency dependence observed with the ester carbonyls primarily reflects hydrogen bonding and the polarity of the immediate vicinity. Conformational changes are accounted for in terms of frequency shifts if the conformational change involves the disposition of the C=O groups and in turn the hydrogen bonding properties. The infrared spectra of phospholipids dispersed in aqueous medium in the liquid crystalline state are inconsistent with a simple phospholipid conformation, e.g., with a conformation as found in the single-crystal structure of 1,2-dimyristoyl-sn-phosphatidylcholine and 1,2-dilauroyl-rac-phosphatidylethanolamine. The spectra support the hypothesis proposed earlier (Hauser et al., Biochemistry, 1988) on the basis of existing single-crystal phospholipid structures and NMR evidence. The hypothesis states that several conformations are present in liquid crystalline phospholipid dispersions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The solution structure of the histidine-containing protein (HPr) from Staphylococcus aureus as determined by two-dimensional 1H-NMR spectroscopy

The three-dimensional solution structure of the heat-stable phosphocarrier protein HPr from Staphylococcus aureus was determined from two-dimensional NMR data by restrained molecular dynamics. It consists of a large twisted antiparallel beta-pleated sheet with four strands A, B, C, and D of amino acids 2-7, 34-37, 40-42 and 60-65. Three right-handed helices A, B, C (amino acids 18-27, 47-53 and 71-85) are positioned on top of this sheet. The aromatic ring of His15 is located in a cleft formed by amino acids 12-17 and 55-58, only the nitrogen (N delta 1) atom which can be phosphorylated by enzyme I is exposed to the water. The side chains of Thr12 and Arg17 are located close to the histidine ring. The regulatory serine residue (Ser46) is located in a hydrophobic patch, its hydroxyl group is water-accessible but forms hydrogen bonds with the amide groups of the backbone. The general features of the three-dimensional structure are similar to those found in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis and Streptococcus faecalis.     

Structure of the 13C-enriched O-deacetylated glucuronoxylomannan of Cryptococcus neoformans serotype A determined by NMR spectroscopy

The complete assignment of 1H and 13C chemical shifts for 99% uniformly 13C-labeled O-deacetylated glucuronoxylomannan (GXM) of Cryptococcus neoformans serotype A isolate 9759-Mu-1 was accomplished by the analysis of HCCH-TOCSY and HCCH-COSY spectra. The sequence of the glycosyl residues was determined by a GHMBC experiment using 20% uniformly 13C-labeled GXM; GXM was prepared by a novel procedure that insured the virtual exclusion of adjacent 13C-labeled carbon atoms. For each residue in the GXM of 9759-Mu-1 we determined its linkage position, its anomeric configuration, and its position in the repeating sequence as follows: [sequence: see text]     

Dual recognition of double-stranded DNA by 2'-aminoethoxy-modified oligonucleotides: the solution structure of an intramolecular triplex obtained by NMR spectroscopy

The solution structure of an intramolecular triple helical oligonucleotide has been solved by NMR. The third strand of the pyrimidine x purine x pyrimidine triplex is composed of 2'-aminoethoxy-modified riboses, whereas the remaining part of the nucleic acid is DNA. The structure around the aminoethoxy modification was obtained with the help of selective isotope labeling in conjunction with isotope-editing experiments. Dinucleotide steps and interstrand connectivities, as well as the complete backbone conformation of the triplex, were derived from J-couplings, NOEs, and 31P chemical shifts. The structure of this triplex, solved by distance geometry, explains the extraordinary stability and increase in rate of triplex formation induced by 2'-aminoethoxy-modified oligonucleotides: apart from the formation of seven base triples, a well-defined hydrogen-bonding network is formed across the Crick-Hoogsteen groove involving the amino protons of the aminoethoxy moieties and the phosphates of the purine strand of the DNA. The modified strand adopts a conformation which is close to an A-type helix, whereas the DNA duplex conformation is best described as an unwound B-type helix. The groove dimensions and helical parameters of the 2'-aminoethoxy-modified rY x dRdY triplex are surprisingly well conserved in comparison with DNA triplexes.     

Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha3 beta2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha3 beta2 interface. Here we describe the three-dimensional solution structure of MII determined using 2D 1H NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 A for the backbone atoms and 0.34 A for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha3beta2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.     

Influence of bulky side chains of amino acids on the solution conformation of peptide fragment (81-92) derivatives of CD4, TYICEVEDQKEE, as studied by NMR spectroscopy and molecular modeling

Solution conformation of CD4 fragment 81-92 TYICEVEDQKEE and two of its benzylated analogues was determined by two-dimensional 1H NMR spectroscopy, distance geometry and simulated annealing techniques. The structures of both benzylated derivatives are similar but are distinct from that of wild-type dodecapeptide. It is concluded from structural analysis that bulky side chain(s) of amino acid(s) at an appropriate position can have a marked effect on the conformation and thus the functions of a peptide.     

Bioactive conformation of a potent stromelysin inhibitor determined by X-nucleus filtered and multidimensional NMR spectroscopy

The biologically active conformation of a novel, very potent, nonpeptidic stromelysin inhibitor was determined by X-nucleus filtered and multidimensional NMR spectroscopy. This bound conformer was subsequently docked into the stromelysin catalytic domain (SCD) using intermolecular distance constraints derived from NOE data. The complex showed the S1' pocket of stromelysin to be the major site of enzyme-inhibitor interaction with other portions of the inhibitor spanning the S2' and S1 binding sites. Theoretical predictions of SCD-inhibitor binding from molecular modeling studies were consistent with the NMR data. Comparison of modeled enzyme-inhibitor complexes for stromelysin and collagenase revealed an alternate binding mode for the inhibitor in collagenase, suggesting a similar binding interaction might also be possible for stromelysin. The NMR results, however, revealed a single SCD-inhibitor binding mode and provided a structural template for the design of more potent stromelysin inhibitors.     

Dexamethasone and dexamethasone phosphate detected by 1H and 19F NMR spectroscopy in the aqueous humour

To apply nuclear magnetic resonance (NMR) spectroscopy to study the penetration of dexamethasone phosphate into the aqueous humour from rabbit following topical administration. After topical administration of 0.1%, 1.0% and 10% dexamethasone phosphate solutions, respectively, samples of aqueous humour were aspirated, freeze-dried, redissolved in deuterium oxide and analyzed by high resolution 1H and 19F NMR spectroscopy. In order to study the lipophilic and hydrophilic metabolites of the drug, samples obtained after application of 1% dexamethasone phosphate were extracted with methanol/chloroform, and then extracted with perchloric acid. In all samples obtained from eyes denuded of the corneal epithelium prior to administration of dexamethasone, signals corresponding to the chemical shifts of the drug were identified in 19F NMR spectra. In the experiments performed with 1% dexamethasone phosphate, both dexamethasone and dexamethasone phosphate were detected in the aqueous humour. Using 10% dexamethasone phosphate solutions, signals from the drug were detected in 1H NMR spectra simultaneously with signals from about twenty other substances present in the aqueous humour. NMR spectroscopy appears to be a valuable method for studying dexamethasone metabolism and penetration into ocular tissues. It provides simultaneous detection of both the drug metabolites and other substances in the sample and might offer a complementary approach to other analytical methods.     

Structures of oligonucleotides containing 5-(methoxymethyl)-2'-deoxyuridine determined by NMR spectroscopy

Base pairing of 5-(methoxymethyl)-2'-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU.A base pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH2 and CH2-O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU.G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.     

Detection of an anhydride intermediate in the carboxypeptidase A catalyzed hydrolysis of a peptide substrate by solid state NMR spectroscopy and its mechanistic implication

We have detected an anhydride intermediate in the CPA catalyzed proteolytic reaction of Gly-Tyr. It appears that since the zinc-bound water molecule which is believed to attack the scissile amide carbonyl carbon in the hydrolysis reaction is excluded by the N-terminal amino group of Gly-Tyr, the carboxylate of Glu-270 becomes to attack the amide bond to generate the anhydride intermediate.     

Conformational studies by NMR of the antimicrobial peptide, drosocin, and its non-glycosylated derivative: effects of glycosylation on solution conformation

Drosocin is a cationic 19 amino acid peptide secreted by Drosophila in response to septic injury. The sequence (GKPRPYSPRPTSHPRPIRV) contains six Pro and four Arg residues which are incorporated into three repeated triplet sequences Pro-Arg-Pro. The peptide is glycosylated at Thr11 and has potent antimicrobial activity. This activity is markedly reduced on deglycosylation, but a structural basis for this has not been previously established. In the current study, the solution conformations of drosocin and its non-glycosylated derivative were determined by NMR spectroscopy and structure calculations. The NMR and structure studies showed that the peptides have significant populations of essentially random coil conformations in aqueous solution. Addition of 50% trifluoroethanol causes the development of small populations of folded conformations, mainly in the form of turns. In particular, turn elements occur near residues 4-7, 10-13, 17, and 18. No substantial difference was detected in the predominantly random coil conformation of the glycosylated and non-glycosylated forms, but there are subtle differences in the small populations of folded conformers. In particular, the turn at residues 10-13 tends toward a more extended structure on glycosylation, while there is some tightening of the downstream turn at residues 17 and 18. There are a significant number of nuclear Overhauser enhancement contacts between the sugar moiety and the peptide near the glycosylation site, consistent with a close association between them. Despite this close association, the pKa of H13, which is proximate to the glycosylation site, was found to be unaffected by glycosylation.     

Comparative conformational analyses of mu-selective dermorphin and delta-selective deltorphin-II in aqueous solution by 1H-NMR spectroscopy

Two-dimensional 1H-NMR methods have been used to obtain complete proton resonance assignments and possible solution conformations of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and deltorphin-II (H-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2), naturally occurring mu- and delta-selective opioids, respectively, in order to examine the conformational characteristics that are closely related to the selectivities towards mu/delta-opioid receptors. With the use of the proton-proton distances derived from ROESY measurements in aqueous solution, 50 possible 3D structures are generated by means of distance geometry calculations. The conformers which satisfy the distance constraints and the torsion angles estimated from JNHC alpha H vicinal coupling constants within the allowable range are then subjected to molecular dynamics simulations for 10 ps after equilibration. Although dermorphin and deltorphin-II are both in equilibrium among many flexible conformers, some conformational differences are observed between these peptides: many conformers of dermorphin show a structure rounded at the N-terminal Tyr-D-Ala-Phe-Gly-Tyr and C-terminal Gly-Tyr-Pro-Ser-NH2 moieties, which are almost at right angles to each other, while those of deltorphin-II are characterized by a 'hook'-shaped backbone structure in which the nearly extended conformation of the Val-Val-Gly-NH2 sequence is located under the folded conformation of the N-terminal Tyr-D-Ala-Phe-Glu sequence. The possible relationship between these conformational characteristics and the mu/delta-opioid receptor selectivities is discussed.     

Interaction and orientation of an alpha-aminoisobutyric acid- and tryptophan-containing short helical peptide pore-former in phospholipid vesicles, as revealed by fluorescence spectroscopy

The interaction and orientation of a membrane protein ion channel model, an alpha-aminoisobutyric acid analogue of gramicidin B (GBA), in egg yolk phosphatidylcholine vesicles was studied by means of fluorescence spectroscopic techniques. GBA helices form stable ion-conducting pores in membranes [Jelokhani-Niaraki et al. (1995) J. Chem. Soc. Perkin Trans. 2, 801-808]. In an alpha-helical model for the peptide, all Trp residues (intrinsic fluorophores) are distributed near the C-terminus. Fluorescence quenching experiments revealed the exposure of the helical peptides' C-termini to aqueous environments. Dansyl-labeled vesicles were used to investigate the GBA dynamism of the interaction with membranes. It was shown that considerable amounts of peptide reside on and in the vicinity of the outer surface of lipid bilayers. The transmembrane transfer to the inner layer is slow due to the high affinity of Trp residues for bilayer interfaces which anchor the peptide to the outer surface. A structural-functional interpretation of the GBA interaction with membranes is presented.