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A S Edison

Publications and source records attributed to A S Edison.

At least 19 recordsLinked to original sources
PubMed2004

An eight-coil high-frequency probehead design for high-throughput nuclear magnetic resonance spectroscopy.

In order to increase the throughput of high-resolution nuclear magnetic resonance spectroscopy a multiple-coil probe, which enables the simultaneous analysis of eight different samples, was designed. The probe, consisting of eight identical solenoidal coils, was constructed for operation at 600 MHz. By using four receivers and radiofrequency switches, spectra from eight different chemical solutions were acquired in the time normally required for one. Two-dimensional COSY, gradient COSY, and TOCSY data have been acquired. Intercoil electrical isolation was between 25 and 45 dB, with signal cross-talk between approximately 1 and 5% measured by NMR. The spectral linewidths for the eight coils were between 3 and 6Hz for a single optimized shim setting.

Equipment Design↗
PubMed2001

Novel agouti-related-protein-based melanocortin-1 receptor antagonist.

The melanocortin receptors are G-protein coupled receptors (GPCRs) that activate the cAMP signal transduction pathway and are stimulated by the melanocortin agonist alpha-melanocyte stimulating hormone (alpha-MSH). Members of these melanocortin receptors are antagonized by agouti (ASP) and agouti-related protein (AGRP), which are the only known endogenous antagonists of GPCRs identified to date. Structure-function studies of the hAGRP(109-118) decapeptide, Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr-NH(2), by replacing the 26-membered disulfide Cys(2)-Cys(9) ring with lactam bridges resulted in the identification of a novel peripheral skin melanocortin-1 receptor (MC1R) antagonist. This antagonist, Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2), possesses a 27-membered ring with the lactam bridge being formed from the Calpha-carboxyl moiety of Glu (instead of the typical side chain carboxyl moiety) with the amine of the diaminopropionic acid (Dpr) residue. This mouse MC1 receptor antagonist (pA(2) = 5.9) is also an antagonist at the brain melanocortin-4 receptor (pA(2) = 6.9), with no observable pharmacology at the melanocortin-3 or -5 receptors. This MC1R hAGRP(109-118) based decapeptide is novel in that AGRP(83-132) itself does not bind to, agonize, or antagonize the skin MC1R. Structural analysis has been performed using two-dimensional (1)H NMR and computer-assisted molecular modeling (CAMM) techniques in attempts to identify structural features of this Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2) (cyclo Glu alphaCOOH-Dpr betaNH) peptide that can differentially result in antagonist versus agonist properties at the mMC1R.

Agouti Signaling Protein↗
PubMed2001

Structural studies of a neuropeptide precursor protein with an RGD proteolytic site.

The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.

Amino Acid Sequence↗
PubMed2001

Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain.

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

Actins↗
PubMed2000

Computational analysis of two similar neuropeptides yields distinct conformational ensembles.

Conformational states and thermodynamic properties for two similar neuropeptides, GDPFLRF-NH(2) and GYPFLRF-NH(2), have been computed by Monte Carlo simulated annealing (MCSA) conformational searches and Metropolis Monte Carlo (MMC) calculations. These peptides were recently shown to have dramatically different conformations in solution by NMR [Edison et al., J Neuroscience 1999;19:6318-6326]. Final conformations of multiple independent MCSA runs were the starting points for MMC calculations, and conformations saved at intervals during MMC runs were characterized in terms of total energy, configuration entropy, side-chain fraction population, and ensemble average inter-nuclear distances. Without the use of any NMR data-generated pseudo-potentials, the present calculations were in excellent qualitative agreement with all previous NMR experimental data and provided a foundation by which to more quantitatively interpret the experimental NMR results. Proteins 2000;40:367-377.

Computer Simulation↗
PubMed2000

Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics.

The primary structure of the Streptococcus mutans lantibiotic mutacin 1140 was elucidated by NMR spectroscopy, mass spectrometry, and chemical sequencing. The structure is in agreement with other closely related lantibiotics, such as epidermin. A novel method was developed in which mutacin 1140 was chemically modified with sodium borohydride followed by ethanethiol, allowing the differentiation of the thioether-containing residues from the dehydrated residues. This double-labeling strategy provides a simple method to reliably identify all modified lantibiotic residues with a minimal amount of material. While NMR spectroscopy is still required to obtain thioether bridging patterns and thus the complete covalent structure, the double-labeling technique, along with mass spectrometry, provides most of the information in a fraction of the time required for a complete NMR analysis. Thus, with these new techniques lantibiotics can be rapidly characterized.

Amino Acid Sequence↗
PubMed1999

Phosphorylation-dependent conformational changes induce a switch in the actin-binding function of MARCKS.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) by protein kinase C eliminates actin filament cross-linking activity, but residual filament binding activity docks phosphorylated MARCKS on filamentous actin. The postulated actin-binding region of MARCKS, which includes a Ca(2+)-calmodulin-binding site, has been portrayed with alpha-helical structure, analogous to other calmodulin-binding domains. Previous speculation suggested that MARCKS may dimerize to form the two functional actin-binding sites requisite for cross-linking activity. Contrary to these hypotheses, we show that MARCKS peptide with actin-cross-linking activity has an extended structure in aqueous solution but assumes a more compact structure upon phosphorylation. We hypothesize that structural changes in the MARCKS peptide induced by phosphorylation create a dynamic structure that, on average, has only one actin-binding site. Moreover, independent of the state of phosphorylation, this peptide is monomeric rather than dimeric, implying that two distinct actin-binding sites are responsible for the actin-cross-linking activity of unphosphorylated MARCKS. These studies uniquely elucidate the mechanism by which phosphorylation of MARCKS induces structural changes and suggest how these structural changes determine biological activity.

Amino Acid Sequence↗
PubMed1999

Conformational ensembles: the role of neuropeptide structures in receptor binding.

Conformational properties of several similar FMRFamide-like neuropeptides from mollusks were investigated by nuclear magnetic resonance (NMR) spectroscopy. It was found that amino acid substitutions in the N-terminal variable regions of the peptides had dramatic effects on the populations of reverse turns in solution. The populations of turns, as measured by two independent NMR parameters, were found to be highly correlated (r(2) = 0.93 and 0. 82) with IC(50) values using receptor membrane preparations from Helix aspersa (Payza, 1987; Payza et al., 1989). These results suggest that the amount of turn in the free peptide can influence the receptor binding affinities of that peptide. On the basis of these observations, a model was developed in which only a single species from a conformational ensemble of an unbound peptide will bind to a particular receptor. Thus, the conformational ensemble reduces the effective concentration of a particular peptide with respect to a particular receptor.

Amino Acid Sequence↗
PubMed1998

Formation of the b subunit dimer is necessary for interaction with F1-ATPase.

In earlier work, we [McCormick, K. A., et al. (1993) J. Biol. Chem. 268, 24683-24691] observed that mutations at Ala-79 of the b subunit affect assembly of F1F0 ATP synthase. Polypeptides modeled on the soluble portion of the b subunit (bsol) with substitutions at the position corresponding to Ala-79 have been used to investigate secondary structure and dimerization of the b subunit. Circular dichroism spectra and chymotrypsin digestion experiments suggested that the recombinant polypeptides with Ala-79 substitutions assumed conformations similar to the bsol polypeptide. However, cross-linking studies of the Ala-79 substitution bsol polypeptides revealed defects in dimerization. The efficiency of dimer formation appeared to be related to the capacity of the altered bsol polypeptides for competing with F1-ATPase for binding to F1-depleted membrane vesicles. Ala-79 substitution polypeptides displaying limited dimerization, such as bsol Ala-79-->Leu, were shown to elute with F1-ATPase during size exclusion chromatography, suggesting a specific interaction. Sedimentation equilibrium studies indicated that 8% of the bsol Ala-79-->Leu polypeptide was in the form of a 30.6 kDa dimer and 92% a 15.3 kDa monomer. When the dimer concentration of bsol Ala-79-->Leu was normalized to the concentration of bsol, both had virtually identical capacities for competing with F1-depleted membrane vesicles for binding F1-ATPase. The result indicated that the amount of dimer formed is directly proportional to its ability to bind F1-ATPase. This suggests that formation of the b subunit dimer may be a necessary step preceding F1-ATPase binding in the assembly of the enzyme complex.

Amino Acid Substitution↗
PubMed1997

afp-1: a gene encoding multiple transcripts of a new class of FMRFamide-like neuropeptides in the nematode Ascaris suum.

We have identified a gene, afp-1, that encodes a new subfamily of six FMRFamide-like neuropeptides in the nematode Ascaris suum. The predicted peptides share the C-terminal sequence PGVLRF-NH2 but have different N-terminal extensions. We discuss possible functional roles of these different peptides based upon experiments with Ascaris as well as results from other organisms. Three of the peptides were previously isolated from extracts of A. suum (4) and three other are novel sequences. The translated product of afp-1 is a precursor protein containing two main halves: a C-terminal region containing a series of putative peptides separated by characteristic processing sites and a relatively hydrophobic N-terminal region with no obvious peptides. Although the overall structure of the translated product of afp-1 is similar to flp-1 from C. elegans (18), there is little evidence for homology between the two nematode neuropeptide genes. At least four different transcripts of afp-1 have been identified. These transcripts differ in their 3' and 5' untranslated regions, and one of the transcripts predicts a truncated precursor protein which contains only the C-terminal peptide-containing region.

Amino Acid Sequence↗
PubMed1994

Calculations of one-, two- and three-bond nuclear spin-spin couplings in a model peptide and correlations with experimental data.

We present ab initio calculations of the Fermi contact term and experimental correlations of six coupling constants, 3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N, 2JC alpha N and 1JC'N, in a peptide as functions of the backbone dihedral angles, phi and psi. Given estimates of experimental uncertainties, we find semiquantitative experimental correlations for 3JHNH alpha, 1JC alpha N and 2JC alpha N, qualitative correlations for 1JC alpha H alpha and 2JC'H alpha, but no experimental correlations of practical utility for 1JC'N, owing to its complex dependence on at least four dihedral angles. Errors in the estimation of dihedral angles from X-ray crystallographic data for proteins, which result from uncertainties in atom-to-atom distances, place substantial limitations on the quantitative reliability of coupling constant calculations fitted to such data. In the accompanying paper [Edison, A.S. et al., J. Biomol. NMR, 4, 543-551] we apply the results of the coupling constant calculations presented here to the estimation of phi and psi angles in staphylococcal nuclease from experimental coupling constants.

Alanine↗
PubMed1994

Estimates of phi and psi torsion angles in proteins from one-, two- and three-bond nuclear spin-spin couplings: application to staphylococcal nuclease.

Calculated coupling constants (3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N and 2JC alpha N) from our accompanying paper [Edison, A.S. et al. (1994) J. Biomol. NMR, 4, 519-542] have been used to generate error surfaces that can provide estimates of the phi and psi angles in proteins. We have used experimental coupling data [3JHNH alpha: Kay, L.E. et al. (1989) J. Am. Chem. Soc., 111, 5488-5490; 1JC alpha H alpha: Vuister, G. W. et al. (1993) J. Biomol. NMR, 3, 67-80; 2JC'H alpha: Vuister, G.W. and Bax, A. (1992) J. Biomol. NMR, 2, 401-405; 1JC alpha N and 2JC alpha N: Delaglio, F. et al. (1991) J. Biomol. NMR, 1, 439-446] to create error surfaces for selected residues of the protein staphylococcal nuclease. The residues were chosen to include all those with five experimental couplings, as well as some with four experimental couplings, to demonstrate the relative importance of 3JHNH alpha and 1JC alpha H alpha. For most of the cases, we obtained good agreement between the X-ray structure [Loll, P.J. and Lattman, E.E. (1989) Protein Struct. Funct. Genet., 5, 183-201] and the NMR data.

Magnetic Resonance Spectroscopy↗
PubMed1994

Inductive effects on the structure of proline residues.

4(S)-Hydroxyproline (Hyp) residues constitute about 10% of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)- fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation, which is the minor isomer in solution, and the pyrrolidine ring of 1 had C gamma-endo pucker. In crystalline 2 and 3 the amide bonds were in the trans conformation, and the pyrrolidine rings had C-exo pucker. The lengths of the bonds between sp3-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1,2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1 < 2 < 3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.

Animals↗
PubMed1993

Mechanism of adenylate kinase. 1H, 13C, and 15N NMR assignments, secondary structures, and substrate binding sites.

Backbone 1H, 13C, and 15N NMR assignments were obtained for the complex of chicken muscle adenylate kinase (AK) with its bisubstrate analog, MgAP5A [magnesium P1,P5-bis(5'-adenosyl)-pentaphosphate]. The assignments were used to elucidate the secondary structures and the enzyme-MgAP5A interactions. The work involves two unusual features: the molecular weight of AK (21.6 kDa) is one of the largest, on a monomeric basis, for which nearly complete assignment has been reported to date, and the assignment was performed at pH 7.1 instead of the acidic pH used for most other proteins. The results are summarized as follows. Firstly, unambiguous sequential assignments of backbone resonances have been achieved effectively by the combined use of two sequential assignment methods: NOE-directed assignments and the recently developed 1J-coupling-directed assignments. The starting points of the assignments were provided by several specifically labeled enzyme samples. Over 90% of the backbone 1H, 13C, and 15N resonances have been assigned. Secondly, spin system information was obtained from the HCCH-TOCSY and HCCH-COSY experiments as well as from 2D homonuclear NMR data. Overall, the side-chain resonances of ca. 40% of the residues, including most of the those displaying NOEs with the adenosine moieties of MgAP5A, have been assigned. Thirdly, secondary structural elements in the AK-MgAP5A complex were identified by extensive analyses of 1H-15N 2D HMQC-NOESY and 3D NOESY-HMQC spectra. Overall, the enzyme consists of ca. 60% alpha-helices and a five-stranded parallel beta-sheet. The results are compared with the secondary structure of the free AK from porcine muscle in crystals [Dreusicke, D., Karplus, P. A., & Schulz, G. E. (1988) J. Mol. Biol. 199, 359-371]. Lastly, most of the intermolecular NOEs between AK and the adenosine moieties of MgAP5A have been identified: Thr39, Leu43, Gly64, Leu66, Val67, Val72, and Gln101 are in proximity to the adenosine moiety of the adenosine 5'-monophosphate site, whereas Thr23 is in proximity to that of the adenosine 5'-triphosphate site. These data are discussed in relation to previous results from site-directed mutagenesis, NMR, and X-ray studies and in relation to the mechanism of catalysis.

Adenylate Kinase↗
PubMed1993

13C alpha-NMR assignments of melittin in methanol and chemical shift correlations with secondary structure.

Melittin is a naturally occurring hexacosa peptide which forms an amphiphilic helix in methanol, a random coil in water, and a tetramer of helices at basic pH or in the presence of a high salt concentration. The monomeric structure in methanol has been well characterized by proton NMR (Pastore et al. (1989) Eur. Biophys. J., 16, 363-367). In the present paper, chemical shifts of the backbone alpha-carbons of melittin in methanol were determined by mapping previously published alpha-proton shifts (Bazzo et al. (1988) Eur. J. Biochem., 173, 139-146), to natural abundance (1H)13C cross peaks appearing in the 2D heteronuclear multiple-quantum NMR spectrum. Changes in chemical shifts consequent to stepwise increases in the percentage of water in a mixed methanol/water solvent system were observed in similar spectra. The alpha-carbon shifts varied more smoothly than the corresponding alpha-proton shifts and were found to correlate with the transition from the helix to the random coil conformer in parallel with changes in the circular dichroism spectrum. Chemical shifts of this peptide are interpreted with regard to the current database of assignments in proteins of known 3D structure (Wishart et al. (1991) J. Mol. Biol., 222, 311-333). The N-terminal region of the peptide shows increased flexibility at lower methanol concentrations, as evidenced by the merger of the alpha-proton resonances of G1 (at 40% and 15% methanol) and G3 (at 15% methanol). Conformational exchange rates for G1 and G3 were estimated by comparison of the experimental spectra with simulated spectra and found to be as large as 4000 s-1 for G1 in 40% and 15% methanol and 600 s-1 for G3 in 15% methanol. Overall, these 1H and 13C chemical shift data support the description of monomeric melittin in methanol currently evolving in the literature and suggest a structure composed of a linked pair of helices with different structural stabilities, each of which experiences dynamical fraying at its free terminus.

Calcium Isotopes↗