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I D Kerr

Publications and source records attributed to I D Kerr.

16 recordsLinked to original sources
PubMed1995

Ion channel formation by synthetic analogues of staphylococcal delta-toxin.

Ion channel formation by three analogues of staphylococcal delta-toxin, an amphipathic and alpha-helical channel-forming peptide, has been evaluated by measurement of ionic currents across planar lipid bilayers. Replacement of beta-branched, hydrophobic residues by leucine and movement of a tryptophan residue from the hydrophilic to the hydrophobic face of the helix does not significantly alter ion channel activity. Removal of the N-terminal blocking group combined with the substitution of glycine-10 by leucine changes the single channel properties of delta-toxin, without altering macroscopic conductance/voltage behaviour. Truncation of the N-terminus by three residues results in complete loss of channel-forming activity. These changes in channel-forming properties upon altering the peptide sequence do not mirror changes in haemolytic activity. The results lend support to the proposal that channel formation and haemolysis are distinct events. Channel properties are discussed in the context of a model in which the pore is formed by a bundle of approximately parallel transbilayer helices.

Amino Acid Sequence↗
PubMed1995

Packing interactions of Aib-containing helices: molecular modeling of parallel dimers of simple hydrophobic helices and of alamethicin.

alpha-Aminoisobutyric acid (Aib) is a helicogenic alpha, alpha-dimethyl amino acid found in channel-forming peptaibols such as alamethicin. Possible effects of Aib on helix-helix packing are analyzed. Simulated annealing via restrained molecular dynamics is used to generate ensembles of approximately parallel helix dimers. Analysis of variations in geometrical and energetic parameters within ensembles defines how tightly a pair of helices interact. Simple hydrophobic helix dimers are compared: Ala20, Leu20, Aib20, and P20, the latter a simple channel-forming peptide [G. Menestrina, K.P. Voges, G. Jung, and G. Boheim (1986) Journal of Membrane Biology, Vol. 93, pp. 111-132]. Ala20 and Leu20 dimers exhibit well-defined ridges-in-grooves packing with helix crossing angles (omega) of the order of +20 degrees. Aib20 alpha-helix dimers are much more loosely packed, as evidenced by a wide range of omega values and small helix-helix interaction energies. However, when in a 3(10) conformation Aib20 helices pack in three well-defined parallel modes, with omega ca. -15 degrees, +5 degrees, and 10 degrees. Comparison of helix-helix interaction energies suggests that dimerization may favor the 3(10) conformation. P20, with 8 Aib residues, also shows looser packing of alpha-helices. The results of these studies of hydrophobic helix dimers are analyzed in the context of the ridges-in-grooves packing model. Simulations are extended to dimers of alamethicin, and of an alamethicin derivative in which all Aib residues are replaced by Leu. This substitution has little effect on helix-helix packing. Rather, such interactions appear to be sensitive to interactions between polar side chains. Overall, the results suggest that Aib may modulate the packing of simple hydrophobic helices, in favor of looser interactions. For more complex amphipathic helices, interactions between polar side chains may be more critical.

Alamethicin↗
PubMed1995

Seven-helix bundles: molecular modeling via restrained molecular dynamics.

Simulated annealing via restrained molecular dynamics (SA/MD) has been used to model compact bundles of seven approximately (anti)parallel alpha-helices. Seven such helix bundles occur, e.g., in bacteriorhodopsin, in rhodopsin, and in the channel-forming N-terminal domain of Bacillus thuringiensis delta-endotoxin. Two classes of model are considered: (a) those consisting of seven Ala20 peptide chains; and (b) those containing a single polypeptide chain, made up of seven Ala20 helices linked by GlyN interhelix loops (where N = 5 or 10). Three different starting C alpha templates for SA/MD are used, in which the seven helices are arranged (a) on a left-handed circular template, (b) on a bacteriorhodopsin-like template, or (c) on a zig-zag template. The ensembles of models generated by SA/MD are analyzed in terms of their geometry and energetics, and the most stable structures from each ensemble are examined in greater detail. Structures resembling bacteriorhodopsin and structures resembling delta-endotoxin are both represented among the most stable structures. delta-Endotoxin-like structures arise from both circular and bacteriorhodopsin-like C alpha templates. A third helix-packing mode occurs several times among the stable structures, regardless of the C alpha template and of the presence or absence of interhelix loops. It is characterized by a "4 + 1" core, in which four helices form a distorted left-handed supercoil around a central, buried helix. The remaining two helices pack onto the outside of the core. This packing mode is comparable with that proposed for rhodopsin on the basis of two-dimensional electron crystallographic and sequence analysis studies.

Amino Acid Sequence↗
PubMed1995

Modelling membrane proteins using structural restraints.

Here we present a procedure for modelling membrane proteins which employs molecular dynamics simulations incorporating target restraints derived from low-resolution structures alongside distance restraints derived from mutagenesis data. The application of the modelling procedure to the closed conformation of the pore domain of the nicotinic acetylcholine receptor is described. This domain is formed by a parallel bundle of five M2 helices. Each M2 helix is kinked due to cumulative distortions of backbone (phi, psi) values. The central region of M2 may adopt a more distorted conformation. This would enable a ring of conserved leucine residues (one from each M2 helix) to pack together, occluding the central pore and thus preventing ion permeation. Molecular dynamics simulations on isolated helices that kink formation is not an inherent property of M2.

Amino Acid Sequence↗
PubMed1994

The alpha-5 segment of Bacillus thuringiensis delta-endotoxin: in vitro activity, ion channel formation and molecular modelling.

A peptide with a sequence corresponding to the highly conserved alpha-5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3429-3436]), was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of alpha-5 at either the N-terminal amino acid or the epsilon-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled alpha-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled alpha-5 peptides were protected from enzymic proteolysis. Functional characterization of alpha-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of alpha-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by alpha-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for alpha-5 in the toxic mechanism of delta-endotoxins, and assign alpha-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alpha-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS Microbiol. Lett. 111, 255-262].

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Alamethicin pyromellitate: an ion-activated channel-forming peptide.

The synthesis and characterization of alamethicin pyromellitate (Alm-PM), a derivative of the channel-forming peptide alamethicin bearing three negative charges at the C-terminus, is described. The self-association of Alm-PM in small unilamellar vesicles of dioleoylphosphatidylcholine (DOPC), monitored using circular dichroism (CD) spectroscopy, occurs much less readily than the self-association of unmodified alamethicin. Channel formation by Alm-PM also occurs less readily and exhibits a higher voltage threshold for activation in planar lipid bilayers and in lipid vesicles. An increase in the salt concentration, and particularly the addition of calcium ions, promotes Alm-PM self-association as monitored by CD spectroscopy. Calcium also facilitates channel formation by Alm-PM both in planar lipid bilayers and in lipid vesicles by lowering the voltage threshold for activation. Thus Alm-PM behaves as an ion-activated ion channel. These results indicate that the self-association of alamethicin-like peptides in membranes is critical for channel formation and that transmembrane flip-flop of peptide helices is not required. In addition, these results demonstrate that the activity of channel-forming peptides may be controlled by controlling the process of self-association.

Alamethicin↗
PubMed1994

Parallel helix bundles and ion channels: molecular modeling via simulated annealing and restrained molecular dynamics.

A parallel bundle of transmembrane (TM) alpha-helices surrounding a central pore is present in several classes of ion channel, including the nicotinic acetylcholine receptor (nAChR). We have modeled bundles of hydrophobic and of amphipathic helices using simulated annealing via restrained molecular dynamics. Bundles of Ala20 helices, with N = 4, 5, or 6 helices/bundle were generated. For all three N values the helices formed left-handed coiled coils, with pitches ranging from 160 A (N = 4) to 240 A (N = 6). Pore radius profiles revealed constrictions at residues 3, 6, 10, 13, and 17. A left-handed coiled coil and a similar pattern of pore constrictions were observed for N = 5 bundles of Leu20. In contrast, N = 5 bundles of Ile20 formed right-handed coiled coils, reflecting loosened packing of helices containing beta-branched side chains. Bundles formed by each of two classes of amphipathic helices were examined: (a) M2a, M2b, and M2c derived from sequences of M2 helices of nAChR; and (b) (LSSLLSL)3, a synthetic channel-forming peptide. Both classes of amphipathic helix formed left-handed coiled coils. For (LSSLLSL)3 the pitch of the coil increased as N increased from 4 to 6. The M2c N = 5 helix bundle is discussed in the context of possible models of the pore domain of nAChR.

Amino Acid Sequence↗
PubMed1994

Studies of the pore-forming domain of a voltage-gated potassium channel protein.

Recent mutagenesis studies have identified a stretch of amino acid residues which form the ion-selective pore of the voltage-gated potassium channel. It has been suggested that this sequence of amino acids forms a beta-barrel structure making up the structure of the ion-selective pore [Hartman, H.A., Kirsch, G.E., Drewe, J.A., Taglialatela, M., Joho, R.H. and Brown, A.M. (1991) Science, 251, 942-944; Yellen, G., Jurman, M.E., Abramson, T. and MacKinnon, R. (1991) Science, 251, 939-942; Yool, A.J. and Schwarz, T.L. (1991) Nature, 349, 700-704]. We have synthesized a polypeptide corresponding to this amino acid sequence (residues 431-449 of the ShA potassium channel from Drosophila). A tetrameric version of this sequence was also synthesized by linking together four of these peptides onto a branching lysine core. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy have been used to investigate the structure of these peptides after their reconstitution into lyso phosphatidylcholine micelles and lipid bilayers composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol. The spectroscopic studies show that these peptides are predominantly alpha-helical in these lipid environments. When incorporated into planar lipid bilayers both peptides induce ion channel activity. Molecular modelling studies based upon the propensity of these peptides to form an alpha-helical secondary structure in a hydrophobic environment are described. These results are discussed in the light of recent mutagenesis and binding studies of the Drosophila Shaker potassium ion channel protein.

Amino Acid Sequence↗
PubMed1993

Hydrophilic surface maps of channel-forming peptides: analysis of amphipathic helices.

Ion channels may be formed by bundles of amphipathic alpha-helices aligned parallel to one another and spanning a lipid bilayer membrane, with the hydrophilic faces of the helices lining a central pore. In order to provide insight into the packing of such helices in bundles, a method has been developed to evaluate hydrophilic surface maps of amphipathic alpha-helices and to display these surfaces in a readily interpretable form. The procedure is based upon empirical energy calculations of interactions of a water molecule with an amphipathic alpha-helix. The method has been applied to three channel-forming peptides: Staphylococcal delta-toxin; alamethicin; and a synthetic leucine- and serine-containing peptide. Particular emphasis is placed upon the effects of sidechain conformational flexibility on hydrophilic surface maps. A family of models of the delta-toxin helix is generated by a simulated annealing procedure. The results of hydrophilic surface map analyses provide more exact definition of the centre of the hydrophilic face of amphipathic helices, and of the variation of the position of the centre in response to changes in sidechain conformation. This information is used to define families of preliminary models for a given ion channel, as is illustrated for delta-toxin.

Alamethicin↗
PubMed1993

Influenza virus M2 protein: a molecular modelling study of the ion channel.

The influenza A M2 protein forms cation-selective ion channels which are blocked by the anti-influenza drug amantadine. A molecular model of the M2 channel is presented in which a bundle of four parallel M2 transbilayer helices surrounds a central ion-permeable pore. Analysis of helix amphipathicity was used to aid determination of the orientation of the helices about their long axes. The helices are tilted such that the N-terminal mouth of the pore is wider than the C-terminal mouth. The channel is lined by residues V27, S31 and I42. Residues D24 and D44 are located at opposite mouths of the pore, which is narrowest in the vicinity of I42. Energy profiles for interaction of the channel with Na+, amantadine-H+ and cyclopentylamine-H+ are evaluated. The interaction profile for Na+ exhibits three minima, one at each mouth of the pore, and one in the region of residue S31. The amantadine-H+ profile exhibits a minimum close to S31 and a barrier near residue I42. This provides a molecular model for amantadine-H+ block of M2 channels. The profile for cyclopentylamine-H+ does not exhibit such a barrier. It is predicted that cyclopentylamine-H+ will not act as an M2 channel blocker.

Amantadine↗
PubMed1991

Ion channels formed by amphipathic helical peptides. A molecular modelling study.

Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an alpha-helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K+ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) delta-toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K(+)-channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. delta-Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K(+)-channel interactions. Rotation of W15 about its C beta-C gamma bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating.

Amino Acid Sequence↗
PubMed1991

A selective biotinylated probe for V1a vasopressin receptors.

We have designed and synthesized a biotinylated vasopressin antagonist which is a selective probe for studying the V1a subtype of vasopressin receptor. Initially we synthesized the novel vasopressin analogue d(CH2)5Tyr(Me)2LysNH2(9)AVP (ALVP). Biotinamidocaproate was subsequently coupled to the epsilon-amino group of ALVP to generate the novel biotinylated probe d(CH2)5Tyr(Me)2Lys(N epsilon-biotinamido-caproate)NH2(9)AVP (ALBtnVP). Pharmacological characterization of ALVP and ALBtnVP established that both ligands were high affinity antagonists at V1a receptors, and that both displayed marked V1a/V2 selectivity. The observation that receptor-bound ALBtnVP was bi-functional, and thereby able to bind conjugated derivatives of avidin or streptavidin, allowed ALBtnVP to be utilized as a selective probe for V1a receptors. This strategy allowed the visualization of V1a receptors on the surface of WRK-1 cells and hippocampal neurons, by using streptavidin-gold with electron microscopy and fluorescein-avidin with light microscopy. We conclude that ALBtnVP is a useful probe for V1a receptors.

Amino Acid Sequence↗
PubMed1990

A scientific paradox--explosives that are safe for use in flammable atmospheres.

Explosives used in coal mines have to be efficient in blasting rock and coal but at the same time should not ignite the flammable atmosphere that can sometimes be encountered underground. When used, the explosives are placed in drilled holes and shot in rounds, with a short delay between each shot. Because the explosive is of low power, later shots in the round may fail to detonate but deflagrate instead. Any flammable atmosphere that might be present would then be ignited. This paper describes investigations of the initiation and propagation of detonation in low-power mining explosives. Qualitative studies show how detonation can fail in the vicinity of delay detonators when the explosive is precompressed prior to detonation. Analysis of X-ray photographs of detonation waves propagating in conditions near to failure enables estimates to be made of reaction-zone shapes, densities, pressures, and particle velocities. This information is used to devise a model of the reaction-zone processes, explain why compression of the explosive can lead to detonation failure, and to assess nearness to failure.

Coal Mining↗