Search PubMed⌕ Search

Biomedical subjects

C E Dempsey

Publications and source records attributed to C E Dempsey.

At least 37 records · Page 2Linked to original sources
PubMed1997

Helix bending in alamethicin: molecular dynamics simulations and amide hydrogen exchange in methanol.

Molecular dynamics simulations of alamethicin in methanol were carried out with either a regular alpha-helical conformation or the x-ray crystal structure as starting structures. The structures rapidly converged to a well-defined hydrogen-bonding pattern with mixed alpha-helical and 3(10)-helical hydrogen bonds, consistent with NMR structural characterization, and did not unfold throughout the 1-ns simulation, despite some sizable backbone fluctuations involving reversible breaking of helical hydrogen bonds. Bending of the helical structure around residues Aib10-Aib13 was associated with reversible flips of the peptide bonds involving G11 (Aib10-G11 or G11-L12 peptide bonds), yielding discrete structural states in which the Aib10 carbonyl or (rarely) the G11 carbonyl was oriented away from the peptide helix. These peptide bond reversals could be accommodated without greatly perturbing the adjacent helical structure, and intramolecular hydrogen bonding was generally maintained in bent states through the formation of new (non-alpha or 3[10]) hydrogen bonds with good geometries: G11 NH-V9 CO (inverse gamma turn), Aib13 NH-Aib8 CO (pi-helix) and, rarely, L12 NH- Q7 NH (pi-helix). These observations may reconcile potentially conflicting NMR structural information for alamethicin in methanol, in which evidence for conformational flexibility in the peptide sequence before P14 (G11-Aib13) contrasts with the stability of backbone amide NH groups to exchange with solvent. Similar reversible reorientation of the Thr11-Gly12 peptide bond of melittin is also observed in dynamics simulations in methanol (R. B. Sessions, N. Gibbs, and C. E. Dempsey, submitted). This phenomenon may have some role in the orientation of the peptide carbonyl in solvating the channel lumen in membrane ion channel states of these peptides.

Alamethicin↗
PubMed1996

Conserved secondary structure in the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2) and the fatty acid synthase acyl carrier protein from Escherichia coli.

The acyl carrier protein (ACP) of Streptomyces coelicolor A3(2) functions as a molecular chaperone during the biosynthesis of the polyketide actinorhodin (act). Here we compare structural features of the polyketide synthase (PKS) ACP, determined by two-dimensional 1H-NMR, with the Escherichia coli fatty acid synthase (FAS) ACP. The PKS ACP contains four helices (residues 7-16 [A], 42-53 [B], 62-67 [C], 72-86 [D]), and a large loop (residues 17-41) having no defined secondary structure with the exception of a turn between residues 21 and 24. The act ACP shows 47% sequence similarity with the E. coli FAS ACP and the results demonstrate that the sequence homology is extended to the secondary structure of the proteins.

Acyl Carrier Protein↗
PubMed1996

Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.

Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is either the bulk solution or water within parallel or antiparallel transmembrane arrays of reconstituted alamethicin.

Alamethicin↗
PubMed1994

Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli.

FarR (formerly P30) has been identified as a fatty acid and fatty acyl-CoA responsive DNA-binding protein. It is encoded by the farR gene (g30) in the citric acid cycle gene cluster of E. coli (gltA-sdhCDAB-sucABCD-farR). The amplified FarR protein specifically bound to the farR promoter (PfarR) and exhibited weak binding to the citrate synthase and lipoamide dehydrogenase promoters. Binding at PfarR was abolished by long-chain fatty acids and their CoA thioesters. In DNaseI footprints, FarR binding at PfarR protected two sites, each characterised by two related 10-bp direct repeats. It is suggested that FarR autoregulates farR expression and may modulate citric acid cycle expression in response to long-chain fatty acids.

Acyl Coenzyme A↗
PubMed1994

Direct 1H NMR evidence for conversion of beta-D-cellobiose to cellobionolactone by cellobiose dehydrogenase from Phanerochaete chrysosporium.

The alpha- and beta-anomers of D-cellobiose were resolved by 1H NMR spectroscopy. Addition of cellobiose dehydrogenase purified from the white-rot P. chrysosporium led to selective conversion of beta-D-cellobiose. The product was identical to cellobionolactone as synthesized from Ca-cellobionate. Overnight incubation of the product led to an altered NMR spectrum, which was also obtained by incubation of cellobionolactone. The new spectrum matched that for Ca-cellobionate. The instability of cellobionolactone explains the detection of cellobionic acid as product in earlier studies.

Basidiomycota↗
PubMed1994

The solution structures of the first and second transmembrane-spanning segments of band 3.

We have studied the structures of synthetic peptides which correspond to the proposed first and second membrane-spanning segments of the human red cell anion transporter (band 3). The peptides, which were acetylated at their N-termini and amidated at the C-termini, comprise the 20 amino acids of residues 405-424 and 21 amino acids of residues 436-456 of the human band 3 sequence. The solution structures of the peptides in trifluoroethanol were studied by two-dimensional NMR spectroscopy. Characteristic NOEs were observed indicating that the peptides adopted a predominantly alpha-helical structure in trifluoroethanol solution. Dynamical simulated annealing using the program XPLOR was employed for the structure calculations. The amide exchange rates in trifluoroethanol have also been measured and are consistent with an alpha-helical structure for the peptides.

Acetylation↗
PubMed1992

Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ.

A trapping method combined with high-resolution nuclear magnetic resonance spectroscopy is described for the measurement of hydrogen-deuterium exchange rates for individual amides of polypeptides bound to fully hydrated, dispersed phospholipid bilayers. Exchange rates were measured for 22 of the 24 amide hydrogens of bee venom melittin bound to bilayers composed of egg phosphatidylcholine/phosphatidylserine (88:12, mol/mol) dispersed in 20 mM sodium acetate, pH 4.0. Amides of residues 5-11 and 16-22 had exchange rates suppressed by between 30- and 1000-fold, and the rate suppression exhibited a helical periodicity with amides on the hydrophobic helix face up to 20-fold more stable than those on the hydrophilic face of the helix. These results demonstrate that under the conditions studied melittin adopts a helical conformation with stable helical hydrogen bonds extending to residue 22 and that the helix is oriented with the hydrophobic face directed toward the membrane interior.

Amides↗
PubMed1992

Quantitation of the effects of an internal proline residue on individual hydrogen bond stabilities in an alpha-helix: pH-dependent amide exchange in melittin and [Ala-14]melittin.

pH-Dependent amide exchange rates in methanol have been measured for 21 of the 25 exchangeable backbone amides of [Ala-14]melittin (P14A), a synthetic analogue of bee venom melittin having a Pro to Ala substitution at residue 14. P14A, like melittin, adopts an alpha-helical conformation in methanol. As previously found for melittin [Dempsey, C. E. (1988) Biochemistry 27, 6893], the exchange data could be fit to curves calculated assuming acid and base catalysis by solvent from which residue-specific values of kH and kOMe, the acid- and base-catalyzed exchange rate constants, respectively, were determined. From a comparison of kmin values, where kmin is the minimum exchange rate in the curve defining the amide exchange rate as a function of pH, with those previously obtained from melittin, the relative stabilities of individual helical hydrogen bonds in the two peptides were calculated in terms of equilibrium constants for hydrogen-bond-breaking backbone fluctuations. Replacement of P14 in melittin with Ala results in stabilization of amides in a central turn of helix by up to 36-fold (delta delta GPro-Ala = 9 kJ mol-1) and elsewhere throughout the helix (residues 5-21) by 2-10-fold. These data indicate a cooperative effect of the Pro to Ala substitution on helix stability and allow a qualitative description of the fluctuational properties of the melittin and P14A helices in methanol. The effects of proline on the properties of the melittin helix are compared with previous theoretical and empirical studies on the effect of proline on the structure and stability of alpha-helices.

Alanine↗
PubMed1992

Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.

Specific side-by-side interactions between transmembrane alpha-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane alpha-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane alpha-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the alpha-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.

Amino Acid Sequence↗
PubMed1991

Contribution of proline-14 to the structure and actions of melittin.

The structure and dynamic properties of bee venom melittin and a synthetic analogue, [Ala14]-melittin (melittin P14A), are compared, using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and amide exchange measurements in methanol. P14A is shown to adopt a regular, stable alpha-helical conformation in solution without the flexibility around the Pro-14 residue found in melittin. P14A has twice the hemolytic activity of melittin but is less able to induce voltage-dependent ion conductance in planar bilayers. The results indicate that helix flexibility afforded by the Pro-14 residue promotes the ability of melittin to adopt the transbilayer associates thought to underlie ion translocation.

Alanine↗
PubMed1991

Reversible disc-micellization of dimyristoylphosphatidylcholine bilayers induced by melittin and [Ala-14]melittin.

The properties of melittin and a synthetic analogue, [Ala-14]melittin (P14A), in inducing reversible transitions between vesicles and micelles at the liquid-crystalline to gel phase transition temperature (Tm) in complexes with saturated phosphatidylcholines has been studied by deuterium NMR and freeze-fracture electron microscopy (EM). At concentrations between 3 and 5 mol% relative to lipid, each peptide causes reversible micellization of dimyristoylphosphatidylcholine (DMPC) bilayers when the temperature is lowered below Tm. At concentrations of 5 mol% relative to lipid, the peptides induce macroscopic magnetic orientation of DMPC bilayers at temperatures around the centre of the lipid phase transition; at temperatures a few degrees above Tm, magnetic orientation is lost. These effects suggest a progressive phase separation of peptide and lipid on cooling the complexes through the phase transition, resulting in increased vesicle deformability. The rates of gel phase micellization, and of bilayer reformation from micelles at temperatures above Tm, are decreased by 100-fold in P14A:DMPC complexes compared with melittin: DMPC complexes. Freeze-fracture EM indicates that P14A suppresses the formation of the gel phase in DMPC bilayers at temperatures below Tm. EM observations of the time-dependence of the reformation of bilayers from micelles after incubating P14A:DMPC micellar complexes at temperatures above Tm indicate that micelles fuse to form growing bilayer sheets from which multilamellar vesicles eventually form. The presence of intramembranous particles (IP) on the fracture faces of both melittin: DMPC complexes and P14A:DMPC complexes in the fluid phase indicates that under the conditions of the study (50 mM Tris-HCl (pH 7.5), 5 mM EDTA) the peptides are organized as discrete aggregates that penetrate deeply into the bilayer.

Deuterium↗
PubMed1990

The actions of melittin on membranes.

The molecular mechanisms underlying the various effects of melittin on membranes have not been completely defined and much of the evidence described indicates that different molecular mechanisms may underlie different actions of the peptide. Ideas about the formation of transbilayer aggregates of melittin under the influence of a transbilayer potential, and for bilayer structural perturbation arising from the location of the peptide helix within the head group region of the membrane have been made based on the crystal structure of the peptide, the kinetics and concentration dependence of melittins membrane actions, together with simple ideas about the conformational properties of amphipathic helical peptides and their interactions with membranes. Physical studies of the interaction of melittin with model membranes have been useful in determining the potential of the peptide to adopt different locations, orientations and association states within membranes under different conditions, but the relationship of the results obtained to the actions of melittin in cell membranes or under the influence of a membrane potential are unclear. Experimental definition of the interaction of melittin with more complex membranes, including the erythrocyte membrane or in bilayers under the influence of a transmembrane potential, will require direct study in these membranes. Experiments employing labeled melittins for ESR, NMR or fluorescence experiments are promising both for their sensitivity (ESR and fluorescence) and the ability to focus on the peptide within the background of endogenous proteins within cell membranes. The study of melittin in model membranes has been useful for the development of methodology for determination of membrane protein structures. Despite the structural complexity of integral membrane proteins, it is interesting that in some respects their study be more straightforward, lacking as they do the elusive properties of melittin (and other structurally labile membrane peptides) which limit the possibility of defining their interaction with membranes in terms of a single conformation, location, orientation and association state within the membrane.

Amino Acid Sequence↗
PubMed1990

Anti-inflammatory activity of bee venom peptide 401 (mast cell degranulating peptide) and compound 48/80 results from mast cell degranulation in vivo.

1. The relationship between the anti-inflammatory activity of the bee venom peptide 401 in the carrageenin-induced oedema of the rat hind paw and its mast cell degranulating activity has been reinvestigated. 2. Mast cell degranulation caused by compound 48/80 (10 mg kg-1) or by allergen challenge in rats sensitized to Nippostrongylus brasiliensis also suppressed rat hind paw oedema in the same test. 3. The anti-inflammatory activities of peptide 401 and compound 48/80 were partially suppressed by pretreatment of rats with mepyramine and methysergide, at doses (2.5 mg kg-1) that completely suppressed skin reactions to these mast cell-derived amines. Pretreatment of rats with compound 48/80 also suppressed the apparent anti-inflammatory actions of peptide 401 and of compound 48/80. 4. Injection of peptide 401 together with carrageenin increased the inflammatory response in the rat hind paw. 5. The anti-inflammatory activity of peptide 401 and of compound 48/80 in the carrageenin-induced swelling of the rat hind paw arises from mast cell degranulation in vivo.

Allergens↗
PubMed1989

The dynamic properties of melittin in solution. Investigations by NMR and molecular dynamics.

Molecular dynamics simulations are described for the peptide melittin. The atomic trajectories are calculated both with normal potential energy functions and with additional distance restraints deduced from nuclear Overhauser effects observed in NMR experiments. The results are compared with NMR data on coupling constants and amide exchange rates and with B-factors from X-ray crystallography. The observed correlations between experiment and molecular dynamics simulations suggest a relatively mobile C-terminus and relatively high flexibility around residue 11. It is noted that the high conformational variation around residue 11 is due in part to the presence of a proline at position 14 which results in a "missing" H-bond in the largely alpha-helical structure. It is also noted that a proline is a common feature of many putative membrane spanning helices. A role for such prolines is suggested.

Bee Venoms↗
PubMed1987

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2.

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Bee Venoms↗
PubMed1987

The interaction of amino-deuteromethylated melittin with phospholipid membranes studied by deuterium NMR.

Melittin, deuteromethylated on each of the four amino groups (Gly-1 N alpha and Lys-7, 21, and 23 N epsilon), was prepared by reductive methylation using deuteroformaldehyde and NaBD3CN. Deuterium NMR spectra were obtained for the modified peptide (D-melittin) bound to phospholipid bilayers and erythrocyte ghosts. D-Melittin at 4 mol% (peptide:lipid) induced reversible transitions between extended bilayers and micelles at the phase-transition temperature in dimyristoylphosphatidylcholine (DMPC) bilayers. These changes in lipid morphology did not occur at 1 mol% D-melittin: DMPC and the peptide was highly motionally restricted in gel in gel-phase lipid.

Bee Venoms↗