Biomedical subjects

J T Pelton

Publications and source records attributed to J T Pelton.

At least 19 recordsLinked to original sources

PubMed2000

Spectroscopic methods for analysis of protein secondary structure.

Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.

Animals↗

PubMed1998

Inhibition of human neutrophil elastase. 4. Design, synthesis, X-ray crystallographic analysis, and structure-activity relationships for a series of P2-modified, orally active peptidyl pentafluoroethyl ketones.

A series of P2-modified, orally active peptidic inhibitors of human neutrophil elastase (HNE) are reported. These pentafluoroethyl ketone-based inhibitors were designed using pentafluoroethyl ketone 1 as a model. Rational structural modifications were made at the P3, P2, and activating group (AG) portions of 1 based on structure-activity relationships (SAR) developed from in vitro (measured Ki) data and information provided by modeling studies that docked inhibitor 1 into the active site of HNE. The modeling-based design was corroborated with X-ray crystallographic analysis of the complex between 1 and porcine pancreatic elastase (PPE) and subsequently the complex between 1 and HNE.

Administration, Oral↗

PubMed1998

Evaluation of proposed modes of binding of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-am idino- 2- naphthyl)propanoic acid hydrochloride and some analogs to factor Xa using a comparative molecular field analysis.

The binding mode of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-ami dino-2- naphthyl)propanoic acid hydrochloride (DX-9065a, 4) to Factor Xa is examined using inhibition data for a series of analogs that have a hydrophobic group as well as basic or dibasic functionality. Comparative molecular field analysis is utilized on a series of DX-9065a analogs in a series of proposed alternative binding modes. A quantitative measure is provided that distinguishes between the proposed binding modes that describes 'how well' the binding mode explains the structure-activity relationship or the best 3D QSAR agrees with the crystallographically determined binding mode. The best model is in agreement with recently available data [Brandstetter et al., J. Biol. Chem., 271 (1996) 29988]. The highest statistical correlation occurs with the second basic group accommodated in the vicinity of Glu97 and a hydrophobic group accommodated in the pocket defined by Phe174, Tyr99 and Trp215. Also, the best model arises when the conformation of the Glu97 side chain is modified such that an H-bond interaction is maintained with the inhibitor if possible. The model also shows a tightening of the S1 pocket as is shown in the recent data described above.

Crystallography↗

PubMed1995

Structural requirements for neuropeptide Y in mast cell and G protein activation.

Incubation of neuropeptide Y or its C-terminal fragments with rat peritoneal mast cells resulted in a dose-dependent histamine release. Fragment 18-36 of neuropeptide Y was the most biologically active peptide. EC25 value on rat mast cells was 7.2 +/- 2.2 nM. Neuropeptide Y was also able to induce a flare response after intradermal injection in humans. The histamine releasing effects of neuropeptide Y related peptides were greatly inhibited by pretreatment of rat mast cells with pertussis toxin or benzalkonium chloride. Neuropeptide Y and C-terminal related peptides also stimulated the GTPase activity of purified heterotrimeric G proteins in a dose-dependent manner from 1 to 50 microM. Binding studies with [125I]neuropeptide Y were unable to provide evidence for the presence of specific binding sites on the surface of mast cells. The alpha helical conformation of neuropeptide Y fragments was studied by measuring the circular dichroism spectra. Neuropeptide Y-(18-36) was the smallest fragment having a strong helical conformation. Our results demonstrate that neuropeptide Y activates mast cells through a non-specific process leading to G protein activation.

Adult↗

PubMed1995

Solution conformational dynamics of the C-terminal residues in endothelin-1 and some analogues: a time-resolved fluorescence study.

The rotational relaxation times of the single tryptophan residues in endothelin-1, [Ala1,3,11,15]endothelin-1, human pro-endothelin-1, the linear hexapeptide Ac-His-Leu-Asp-Ile-Ile-Trp which corresponds to the C-terminal residues 16-21 in endothelin-1, the cyclic pentapeptide BQ123, and several di- and tri-peptides possessing C-terminal tryptophan residues have been determined from time-resolved fluorescence anisotropy decays obtained by phase/modulation techniques. Fluorescence lifetime distribution widths have also been examined as predictors of conformational heterogeneity/restriction. A significant contribution from a slow rotational component supports either the persistence, on the nano-second timescale at least, of a non-flexible alpha-helical structure for the C-terminal tail residues of endothelin-1 in water as solvent, as seen in the X-ray crystallographic structure, or the interaction of the C-terminal tail residues 16-21 with the constrained disulfide-bridged core residues 1-15. This slow rotational contribution is less evident in the linear, acyclic tetraalanine analogue but greatly increased in pro-endothelin-1. In BQ123 the fluorescence characteristics support the occurrence of a dominant rotameric form involving the indole sidechain of the D-tryptophan residue (C alpha-C beta torsion angle chi 1 of 60 degrees, as previously determined by NMR.

Amino Acid Sequence↗

PubMed1994

Stimulation of cGMP-dependent protein kinase I alpha by a peptide from its own sequence. An investigation by enzymology, circular dichroism and 1H NMR of the activity and structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide.

The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1994

The receptors for endothelins and their analogues in SK-N-MC neuroblastoma cells.

The potency order of peptides to inhibit [125I]endothelin-1 binding and to stimulate phosphatidylinositol phosphate (PtdInsP) turnover in SK-N-MC cells was consistent with the presence of ETA-endothelin receptors. Divalent cations enhanced [125I]endothelin-1 binding by, in the case of Mn2+, increasing radioligand affinity. Mn2+ did not induce conformational changes in endothelin-1, and its effect was maintained in solubilized receptors. Hence, metal ions may directly interact with endothelin receptors. The effects of BQ-123 and [Ala1,3,11,15]endothelin-1 on PtdInsP turnover were investigated. Concentration-response curves of endothelins were modeled by a second-order equation that assumes pseudoirreversible ligand binding.

Binding, Competitive↗

PubMed1994

Echistatin: the refined structure of a disintegrin in solution by 1H NMR and restrained molecular dynamics.

The structure of the disintegrin echistatin has been determined by 1H NMR, distance geometry calculations and restrained molecular dynamics simulations. The structure has been refined from the preliminary distance geometry calculations with the inclusion of additional 1H NMR data and hydrogen bonds identified in early stages of the molecular dynamics calculations. The calculations reported here allow a distinction to be made between the two possible disulfide bridging patterns-echistatin is crosslinked as follows: Cys2-Cys11, Cys7-Cys32, Cys8-Cys37, Cys20-Cys39. The final set of structures gives an average pairwise root mean square distance of 0.100 nm (calculated over the backbone atoms of residues Ser4-Cys20 and Asp30-Pro40). The core of echistatin is a well defined though irregular structure, composed of a series of non-classical turns crosslinked by the disulfide bridges and stabilised by hydrogen bonds. The RGD sequence is located in a protruding loop whose stem is formed by two rigid, hydrogen-bonded strands (Thr18-Cys20, Asp30-Cys32). The RGD sequence is connected to this structure by short, flexible segments. High (but not unlimited) mobility is probably necessary for fast recognition and fitting to the integrin receptors. Sequence variability among the disintegrins is found in the segments flanking the RGD sequence, suggesting that these may be important in conferring specificity for the receptors.

Amino Acid Sequence↗

PubMed1993

Structural characterization of myo-inositol monophosphatase from bovine brain by secondary structure prediction, fluorescence, circular dichroism and Raman spectroscopy.

Structural aspects of myo-inositol monophosphatase were examined by spectroscopic techniques and empirical prediction methods. The enzyme belongs to the alpha/beta class of proteins, with approx. 33% alpha-helix and 29% beta-sheet, as shown by circular dichroism (CD), Raman spectroscopy and prediction based on the amino-acid sequence. The Raman spectrum also suggests that the three tryptophan residues in myo-inositol monophosphatase are not exposed to solvent. This was confirmed by a blue shift of 25 nm in the fluorescence emission spectrum, as compared to tryptophan in water, and by quenching studies with acrylamide. The enzyme shows a transition temperature of 87 degrees C for the CD signal at 222 nm. This remarkable heat stability is not due to the presence of disulfide bonds, since both the Raman spectrum and chemical modification studies clearly indicate that all six cysteine residues are in the reduced state.

Acrylamide↗

PubMed1993

The structure and specificity of endothelin receptors: their importance in physiology and medicine.

In addition to involvement in vascular endothelium-smooth muscle communication, the secretion of and receptors for, endothelins are widely distributed. Two cloned receptor subtypes are G-protein-coupled to several intracellular messengers, predominantly inositol phosphates. From a knowledge of structure-activity relationships and peptide conformations, details of receptor architecture and selective agents, including nonpeptides and antagonists, have been discovered. From the nature of the actions of endothelins, receptor distributions (including CNS) and plasma levels, it is concluded that they are paracrine factors normally involved in long-term cellular regulation, but which may be important in several pathologies, many of which are stress-related.

Amino Acid Sequence↗

PubMed1993

Endothelins--from receptors to medicine.

Since the discovery of endothelins, peptides with exceptional vasoconstrictor potency that were originally suggested to act by causing the opening of Ca2+ channels, it has emerged that these agents are important in intercellular communication in many tissues. They exert their effects through G protein-coupled receptors, of which two classes have been cloned. Robert Miller, John Pelton and John Huggins review the progress made towards a molecular understanding of ligand recognition by endothelin receptors. Receptor-selective agonists and antagonists have emerged from attempts to understand the three-dimensional structure of the endothelin pharmacophore, from structure-activity studies and from rapid-screening programmes. From the nature of the secretion and action of endothelins, it would seem that these peptides are involved in long-term changes rather than in acute responses to stimuli, and that they are likely to be important in a number of pathological states. Evidence suggests that receptor antagonists with appropriate affinity and selectivity may be useful in the treatment of conditions as diverse as hypertension, ulcerogenesis and ciclosporin toxicity.

Amino Acid Sequence↗

PubMed1992

Conformational studies of the 'RGD' containing peptide echistatin and close analogs by circular dichroism and fluorescence.

Echistatin, one of the smallest and most active natural disintegrins, and its [Trp13]echistatin, [Trp27]echistatin, [Phe13,Trp31]echistatin analogs have been investigated by far-UV circular dichroism spectroscopy and fluorescence spectroscopy. All analogs inhibited ADP-stimulated platelet aggregation with EC50 values between 30 and 50 nM. The analogs were related closely, both in the CD spectral properties, characteristic of turn conformations, and in the location of isodichroic points connected to conformational transitions upon temperature increase. The low fluorescence quantum yield for Trp13 of 0.018, which could be enhanced 2.7-fold by DTT reduction of the peptide, is ascribed to a close proximity of this Trp13 residue to a disulfide bond. Calculation of the efficiency of fluorescence resonance energy transfer (FRET) yielded distances of 11.5 +/- 0.8 A for Tyr31-Trp27 in [Trp27]echistatin, and more than 15 A for Tyr31-Trp13 in [Trp13]echistatin, in good agreement with the structure of echistatin deduced from earlier NMR-molecular modeling studies. Both Trp13 and Trp27 in the respective analogs were quenched effectively by acrylamide with bimolecular quenching constants of 3.36 x 10(9) M-1 s-1 and 3.72 x 10(9) M-1 s-1, respectively. Iodide anion had negligible quenching effect on Trp13, despite high exposure of this residue to water, but was only 2-fold less efficient than acrylamide in quenching Trp27 fluorescence. Steady-state fluorescence anisotropy data, together with mean fluorescence lifetimes of 1.25 ns for Trp13 and 3.84 ns for Trp27 derived from full fluorescence lifetime decay analyses, yielded long rotational relaxation times of 1.39 +/- 0.18 and 1.35 +/- 0.17 ns, respectively, for these residues comparable to the expected overall rotation time of the peptides. The 'RGD'-containing loop appears to be restricted in movement on the nanosecond timescale with respect to the compact core of the peptide.

Amino Acid Sequence↗

PubMed1992

Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy.

Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition, comparison of the far-UV CD spectrum of reconstituted CK-2 with the spectra of the subunits indicates that conformational changes occur in the backbone region upon association. Such changes may explain the increased enzyme activity of the holoenzyme relative to that of the alpha subunit itself. In contrast, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of heparin, presumably by its binding to the polylysine stretch at amino acid positions 74-77. Heat denaturation experiments (25-90 degrees C) support the notion that heparin may provide a local protective function. A similar but much larger effect was also observed in the presence of the beta subunit only, which supports previous suggestions of a protective function for this subunit. These results indicate that the protection provided by the beta subunit and the increased enzyme activity of the holoenzyme may arise, in part, from a stabilization of the conformation of the enzyme complex and an increase in alpha-helical content.

Amino Acid Sequence↗

PubMed1992

Structure of neuropeptide Y dimer in solution.

The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11-36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N-terminus (residues 1-9) appears as an unstructured mobile segment. Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 +/- 0.6 microM at pH 2-8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.

Animals↗

PubMed1992

BQ-123, cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu), is a non-competitive antagonist of the actions of endothelin-1 in SK-N-MC human neuroblastoma cells.

SK-N-MC cells, derived from a human neuroblastoma, respond to endothelin (ET) peptides with an increase in the free intracellular calcium concentration. The response is biphasic, with the secondary plateau phase being abolished or reduced by removal of extracellular Ca2+ or by the presence of 100nM nitrendipine. Restoration of Ca2+ to the bathing solution in cells stimulated by ET-1 in the absence of Ca2+ caused the plateau to reappear. The order of potency of ET family peptides was ET-2 greater than or equal to sarafotoxin S6b greater than or equal to ET-1 much greater than ET-3, suggesting that ETA receptors mediate the response. Sarafotoxin S6c and the C-terminal hexapeptide endothelin (16-21) were inactive in these cells. [Ala1,3,11,15]ET-1, a linear analogue of ET-1 which has been suggested to be a selective ETB receptor agonist, was a weak competitive antagonist of the actions of ET-1 in these cells. However, BQ-123, recently introduced as a selective and competitive antagonist at ETA receptors, was a potent non-competitive antagonist of ET-1 giving a 50% reduction in the maximum response at 6nM.

Calcium↗

PubMed1992

Conformational study of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu], an endothelin-A receptor-selective antagonist.

The conformation of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu], (BQ123), an endothelin-A receptor-selective antagonist, has been studied in 20% acetonitrile in water by CD and NMR spectroscopy. CD studies showed the peptide adopted a similar, constrained conformation in both water alone and 20% acetonitrile in water. NMR spectra showed the proline residue to be in the trans conformation and 2 of the NH protons to exchange slowly with the solvent, indicating hydrogen bonding. Structural constraints derived from the NMR spectra were used to define the conformation in molecular dynamics simulations. A single backbone conformation is observed for the cycle, comprising a beta type II turn and a gamma' turn.

Amino Acid Sequence↗

PubMed1992

Discrimination between ETA- and ETB-receptor-mediated effects of endothelin-1 and [Ala1,3,11,15]endothelin-1 by BQ-123 in the anaesthetized rat.

1. The influence of BQ-123 (a selective ETA-receptor antagonist) on the haemodynamic response elicited by endothelin-1 (ET-1) and [Ala1,3,11,15]ET-1 (a selective ETB-receptor agonist) was studied in anaesthetized rats instrumented with ultrasonic Doppler flow probes on the carotid, coeliac, mesenteric, renal and iliac arteries. 2. BQ-123 alone (1.6 mumol kg-1, i.v.) induced a decrease in femoral mean arterial pressure (AP), accompanied by a systemic vasodilatation. The response was maximal after 3 min and then returned slowly to baseline. None of these effects was observed after a 0.016 mumol kg-1 dose of BQ-123. 3. ET-1 (1 nmol kg-1, i.v.) induced a biphasic response characterized by a transient initial decrease in AP accompanied by regional vasodilatation (mainly in the carotid and iliac beds) and by immediate mesenteric and renal vasoconstrictions. This was followed, within 1 min, by a marked and prolonged increase in AP accompanied by systemic vasoconstriction. Pretreatment with BQ-123 (1.6 mumol kg-1, i.v., 8 min before ET-1) increased and prolonged the vasodilator effect of ET-1 (mainly in the carotid, coeliac, mesenteric and iliac beds) and reduced its systemic vasoconstrictor effects with marked regional differences (the coeliac, mesenteric and renal beds being poorly affected). 4. [Ala1,3,11,15]ET-1 (3 nmol kg-1, i.v.) induced an initial and marked decrease in AP accompanied by regional vasodilatation (mainly in the carotid, coeliac and iliac beds) and by mesenteric and renal vasoconstrictions. This was followed, within 5 min, by a small increase in AP and systemic vasoconstriction. All these effects were dose-dependent. Pretreatment with BQ-123 (1.6 tmol kg'; 8 min before ET-1) did not modify the early effect of [Ala'3""5]ET-l, but abolished its secondary vasoconstrictor effect except in the mesenteric bed.5. This study demonstrates that pretreatment with BQ-123 not only reduced a large part of the sustained vasoconstrictor activity of ET-1, suggesting the involvement of ETA-receptors, but also enhanced the early vasodilator activity of ET-1 revealing a functional antagonism between the two effects. The vasodilator effect of [Ala1"3""l '5]ET-1 was not affected by BQ-123 and ET-1 induced a similar vasodilatation, that was potentiated by BQ-123, suggesting the involvement of ETB-receptors in this vasodilator response. Marked regional differences were however observed which might be partly related to different levels of functional antagonism between ETB- and ETA-mediated effects, but differences in receptor types, or subtypes, cannot be excluded, mainly in the mesenteric and renals beds.

Animals↗

PubMed1991

The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.

Amino Acid Sequence↗