Jason Breed

A novel method for structure-based prediction of ion channel conductance properties.

A rapid and easy-to-use method of predicting the conductance of an ion channel from its three-dimensional structure is presented. The method combines the pore dimensions of the channel as measured in the HOLE program with an Ohmic model of conductance. An empirically based correction factor is then applied. The method yielded good results for six experimental channel structures (none of which were included in the training set) with predictions accurate to within an average factor of 1.62 to the true values. The predictive r2 was equal to 0.90, which is indicative of a good predictive ability. The procedure is used to validate model structures of alamethicin and phospholamban. Two genuine predictions for the conductance of channels with known structure but without reported conductances are given. A modification of the procedure that calculates the expected results for the effect of the addition of nonelectrolyte polymers on conductance is set out. Results for a cholera toxin B-subunit crystal structure agree well with the measured values. The difficulty in interpreting such studies is discussed, with the conclusion that measurements on channels of known structure are required.

Water in channel-like cavities: structure and dynamics.

Ion channels contain narrow columns of water molecules. It is of interest to compare the structure and dynamics of such intrapore water with those of the bulk solvent. Molecular dynamics simulations of modified TIP3P water molecules confined within channel-like cavities have been performed and the orientation and dynamics of the water molecules analyzed. Channels were modeled as cylindrical cavities with lengths ranging from 15 to 60 A and radii from 3 to 12 A. At the end of the molecular dynamics simulations water molecules were observed to be ordered into approximately concentric cylindrical shells. The waters of the outermost shell were oriented such that their dipoles were on average perpendicular to the normal of the wall of the cavity. Water dynamics were analyzed in terms of self-diffusion coefficients and rotational reorientation rates. For cavities of radii 3 and 6 A, water mobility was reduced relative to that of simulated bulk water. For 9- and 12-A radii confined water molecules exhibited mobilities comparable with that of the bulk solvent. If water molecules were confined within an hourglass-shaped cavity (with a central radius of 3 A increasing to 12 A at either end) a gradient of water mobility was observed along the cavity axis. Thus, water within simple models of transbilayer channels exhibits perturbations of structure and dynamics relative to bulk water. In particular the reduction of rotational reorientation rate is expected to alter the local dielectric constant within a transbilayer pore.

Conservation analysis and structure prediction of the SH2 family of phosphotyrosine binding domains.

Src homology 2 (SH2) regions are short (approximately 100 amino acids), non‐catalytic domains conserved among a wide variety of proteins involved in cytoplasmic signaling induced by growth factors. It is thought that SH2 domains play an important role in the intracellular response to growth factor stimulation by binding to phosphotyrosine containing proteins. In this paper we apply the techniques of multiple sequence alignment, secondary structure prediction and conservation analysis to 67 SH2 domain amino acid sciences. This combined approach predicts seven core secondary structure regions with the pattern β‐α‐β‐β‐β‐β‐α, identifies those residues most likely to be buried in the hydrophobic core of the native SH2 domain, and highlights patterns of conservation indicative or secondary structural elements. Residues likely to be involved in phosphotyrosine binding are shown and orientations of the predicted secondary structures suggested which could enable such residues to cooperate in phosphate binding. We propose a consensus pattern that encapsulates the principal conserved features or the SH2 domains. Comparison of the proposed SH2 domain or ak1 to this pattern shows only matches, suggesting that this domain may not exhibit SH2‐like properties.

Molecular dynamics simulations of water within models of ion channels.

The transbilayer pores formed by ion channel proteins contain extended columns of water molecules. The dynamic properties of such waters have been suggested to differ from those of water in its bulk state. Molecular dynamics simulations of ion channel models solvated within and at the mouths of their pores are used to investigate the dynamics and structure of intra-pore water. Three classes of channel model are investigated: a) parallel bundles of hydrophobic (Ala20) alpha-helices; b) eight-stranded hydrophobic (Ala10) antiparallel beta-barrels; and c) parallel bundles of amphipathic alpha-helices (namely, delta-toxin, alamethicin, and nicotinic acetylcholine receptor M2 helix). The self-diffusion coefficients of water molecules within the pores are reduced significantly relative to bulk water in all of the models. Water rotational reorientation rates are also reduced within the pores, particularly in those pores formed by alpha-helix bundles. In the narrowest pore (that of the Ala20 pentameric helix bundle) self-diffusion coefficients and reorientation rates of intra-pore waters are reduced by approximately an order of magnitude relative to bulk solvent. In Ala20 helix bundles the water dipoles orient antiparallel to the helix dipoles. Such dipole/dipole interaction between water and pore may explain how water-filled ion channels may be formed by hydrophobic helices. In the bundles of amphipathic helices the orientation of water dipoles is modulated by the presence of charged side chains. No preferential orientation of water dipoles relative to the pore axis is observed in the hydrophobic beta-barrel models.

Simulation Studies of Alamethicin-Bilayer Interactions

Alamethicin is an alpha-helical peptide that forms voltage-activated ion channels. Experimental data suggest that channel formation occurs via voltage-dependent insertion of alamethicin helices into lipid bilayers, followed by self-assembly of inserted helices to form a parallel helix bundle. Changes in the kink angle of the alamethicin helix about its central proline residue have also been suggested to play a role in channel gating. Alamethicin helices generated by simulated annealing and restrained molecular dynamics adopt a kink angle similar to that in the x-ray crystal structure, even if such simulations start with an idealized unkinked helix. This suggests that the kinked helix represents a stable conformation of the molecule. Molecular dynamics simulations in the presence of a simple bilayer model and a transbilayer voltage difference are used to explore possible mechanisms of helix insertion. The bilayer is represented by a hydrophobicity potential. An alamethicin helix inserts spontaneously in the absence of a transbilayer voltage. Application of a cis positive voltage decreases the time to insertion. The helix kink angle fluctuates during the simulations. Insertion of the helix is associated with a decrease in the mean kink angle, thus helping the alamethicin molecule to span the bilayer. The simulation results are discussed in terms of models of alamethicin channel gating.

The structure of porin from Paracoccus denitrificans at 3.1 A resolution

© 1997 Federation of European Biochemical Societies.

ALAMETHICIN CHANNELS: MODELLING VIA RESTRAINED MOLECULAR DYNAMICS SIMULATIONS

Alamethicin channels have been modelled as approximately parallel bundles of transbilayer helices containing between N = 4 and 8 helices per bundle. Initial models were generated by in vacuo restrained molecular dynamics (MD) simulations, and were refined by 60 ps MD simulations with water molecules present within and at the mouths of the central pore. The helix bundles were stabilized by networks of H-bonds between intra-pore water molecules and Gln-7 side-chains. Channel conductances were predicted on the basis of pore radius profiles, and suggested that the N = 4 bundle formed an occluded pore, whereas pores with N > or = 5 helices per bundle were open. Continuum electrostatics calculations suggested that the N = 6 pore is cation-selective, whereas pores with N > or = 7 helices per bundle were predicted to be somewhat less ion-selective.

Ion channel stability and hydrogen bonding. Molecular modelling of channels formed by synthetic alamethicin analogues.

Several analogues of the channel-forming peptaibol alamethicin have been demonstrated to exhibit faster switching between channel substates than does unmodified alamethicin. Molecular modelling studies are used to explore the possible molecular basis of these differences. Models of channels formed by alamethicin analogues were generated by restrained molecular dynamics in vacuo and refined by short molecular dynamics simulations with water molecules within and at either mouth of the channel. A decrease in backbone solvation was found to correlate with a decrease in open channel stability between alamethicin and an analogue in which all alpha-amino-isobutyric acid residues of alamethicin were replaced by leucine. A decrease in the extent of hydrogen-bonding at residue 7 correlates with lower open channel stabilities of analogues in which the glutamine at position 7 was replaced by smaller polar sidechains. These two observations indicate the importance of alamethicin/water H-bonds in stabilizing the open channel.

Polarity-dependent conformational switching of a peptide mimicking the S4–S5 linker of the voltage-sensitive sodium channel

The S4-S5 linker (or S45) in voltage-sensitive sodium channels was previously shown to be involved in the permeation pathway. The secondary structure, investigated by circular dichroism, of a S4-S45 peptide from domain IV and its fragments (including S45) is reported here and compared with that of the homologous peptide from domain II as a function of the solvent dielectric constant. The reduction in helicity seen for S4-S45 (II) in polar media is cancelled in membrane-like environment. The most striking result-- a sharp alpha-helix --> beta-sheet transition upon exposure of the S45 moiety to aqueous solvents-- is discussed as regards channel activation and selectivity.

Purification, crystallization and preliminary crystallographic analysis of the periplasmic binding protein ProX from Escherichia coli

A periplasmic binding protein (ProX) for the compatible solutes glycine betaine and proline betaine from Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. Crystals were grown using a protein concentration of 10 mg ml−1 and a precipitant of 26–28% PEG 4000 in 50 mM PIPES pH 6.2–6.4. Native diffraction data to 1.93 A resolution have been obtained from crystals at 290 K. The crystals belong to the space group P212121, with unit-cell parameters a = 48.1, b = 55.0, c = 115.7 A, and contain one molecule per asymmetric unit.