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B A Wallace

Publications and source records attributed to B A Wallace.

At least 37 records · Page 2Linked to original sources
PubMed2003

Solution NMR studies of antiamoebin, a membrane channel-forming polypeptide.

Antiamoebin I is a membrane-active peptaibol produced by fungi of the species Emericellopsis which is capable of forming ion channels in membranes. Previous structure determinations by x-ray crystallography have shown the molecule is mostly helical, with a deep bend in the center of the polypeptide, and that the backbone structure is independent of the solvent used for crystallization. In this study, the solution structure of antiamoebin was determined by NMR spectroscopy in methanol, a solvent from which one of the crystal structures was determined. The ensemble of structures produced exhibit a right-handed helical C terminus and a left-handed helical conformation toward the N-terminus, in contrast to the completely right-handed helices found in the crystal structures. The NMR results also suggest that a "hinge" region exists, which gives flexibility to the polypeptide in the central region, and which could have functional implications for the membrane insertion process. A model for the membrane insertion and assembly process is proposed based on the antiamoebin solution and crystal structures, and is contrasted with the assembly and insertion mechanism proposed for other ion channel-forming polypeptides.

Anti-Bacterial Agents↗
PubMed2003

Circular dichroism and synchrotron radiation circular dichroism spectroscopy: tools for drug discovery.

CD spectroscopy is an established and valuable technique for examining protein structure, dynamics and folding. Because of its ability to sensitively detect conformational changes, it has important potential for drug discovery, enabling screening for ligand and drug binding, and detection of potential candidates for new pharmaceuticals. The binding of the anti-tumour agent Taxol to the anti-apoptosis protein Bcl-2 [Rodi, Janes, Sanganee, Holton, Wallace and Makowski (1999) J. Mol. Biol. 285, 197-204] and the binding of the anti-epileptic drug lamotrigine to voltage-gated sodium channels [Cronin, O'Reilly, Duclohier and Wallace (2003) J. Biol. Chem. 278, 10675-10682] are used as examples to show changes detectable by CD involving secondary structure, and are contrasted with the binding of the agonist carbamylcholine to acetylcholine receptors [Mielke and Wallace (1988) J. Biol. Chem. 263, 8177-8182], an example where binding does not involve a secondary structural change. Synchrotron radiation CD spectroscopy offers significant enhancements with respect to conventional CD spectroscopy, which will enable its usage for high-throughput screening and as a tool in 'chemical genomics' or 'reverse chemical genetics' strategies for ligand identification. The lower wavelength data available enable more detailed, sensitive and accurate detection, the higher light intensity permits much smaller amounts of both proteins and drug candidates to be used in the screening, and future technological developments in sample handling and detection should enable automated high-throughput screening to be performed.

Antineoplastic Agents↗
PubMed2003

Analyses of circular dichroism spectra of membrane proteins.

Circular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.

Animals↗
PubMed2003

The temperature dependence of gramicidin conformational States in octanol.

In lipid bilayers and organic solvents, the hydrophobic polypeptide gramicidin adopts a number of different conformations, some of which are capable of conducting monovalent cations across phospholipid membranes. The equilibria between conformations have been shown to be influenced by factors such as lipid chain length, solvent, concentration and salt. In this study, the temperature dependence of the equilibrium mixture of double helical ion-free gramicidin in octanol was examined using circular dichroism spectroscopy.

Amino Acid Sequence↗
PubMed2002

Characterization and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system.

Proteins bearing a signal peptide with a consensus twin-arginine motif are translocated via the Tat pathway, a multiprotein system consisting minimally of the integral inner membrane proteins TatA, TatB, and TatC. On a molar basis, TatA is the major pathway component. Here we show that TatA can be purified independently of the other Tat proteins as a 460 kDa homooligomeric complex. Homooligomer formation requires the amino-terminal membrane-anchoring domain of TatA. According to circular dichroism spectroscopy, approximately half of the TatA polypeptide forms alpha-helical secondary structure in both detergent solution and proteoliposomes. An expressed construct without the transmembrane segment is largely unstructured in aqueous solution but is able to insert into phospholipid monolayers and interacts with membrane bilayers. Protease accessibility experiments indicate that the extramembranous region of TatA is located at the cytoplasmic face of the cell membrane.

Cell Membrane↗
PubMed2002

Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation.

Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.

Amino Acid Sequence↗
PubMed2002

Model for a helical bundle channel based on the high-resolution crystal structure of trichotoxin_A50E.

Trichotoxin_A50E is an 18-residue peptaibol antibiotic which forms multimeric transmembrane channels through self-association. The crystal structure of trichotoxin has been determined at a resolution of 0.9 A. The trichotoxin sequence contains nine helix-promoting Aib residues, which contribute to the formation of an entirely helical structure that has a central bend of 8-10 degrees located between residues 10-13. Trichotoxin is the first solved structure of the peptaibol family that is all alpha-helix as opposed to containing part or all 3(10)-helix. Gln residues in positions 6 and 17 produce a polar face, and are proposed to form the channel lumen. An octameric model channel has been constructed from the crystal structure. It has a central pore of approximately 4-5 A radius, a size sufficient to enable transport of ions, with a constricted region at one end, formed by a ring of Gln6 residues. Electrostatic calculations are consistent with it being a cationic channel.

Acetonitriles↗
PubMed2002

Interactions of the type III secretion pathway proteins LcrV and LcrG from Yersinia pestis are mediated by coiled-coil domains.

The type III secretion system is used by pathogenic Yersinia to translocate virulence factors into the host cell. A key component is the multifunctional LcrV protein, which is present on the bacterial surface prior to host cell contact and up-regulates translocation by blocking the repressive action of the LcrG protein on the cytosolic side of the secretion apparatus. The functions of LcrV are proposed to involve self-interactions (multimerization) and interactions with other proteins including LcrG. Coiled-coil motifs predicted to be present are thought to play a role in mediating these protein-protein interactions. We have purified recombinant LcrV, LcrG, and site-directed mutants of LcrV and demonstrated the structural integrity of these proteins using circular dichroism spectroscopy. We show that LcrV interacts both with itself and with LcrG and have obtained micromolar and nanomolar affinities for these interactions, respectively. The effects of LcrV mutations upon LcrG binding suggest that coiled-coil interactions indeed play a significant role in complex formation. In addition, comparisons of secretion patterns of effector proteins in Yersinia, arising from wild type and mutants of LcrV, support the proposed role of LcrG in titration of LcrV in vivo but also suggest that other factors may be involved.

Amino Acid Sequence↗
PubMed2002

Structure and function of voltage-dependent ion channel regulatory beta subunits.

Voltage-dependent K(+), Ca(2+), and Na(+) channels play vital roles in basic physiological processes, including management of the action potential, signal transduction, and secretion. They share the common function of passively transporting ions across cell membranes; thus, it would not be surprising if they should exhibit similarities of both structure and mechanism. Indeed, the principal pore-forming (alpha) subunits of each show either exact or approximate 4-fold symmetry and share a similar transmembrane topology, and all are gated by changes in membrane potential. Furthermore, these channels all possess an auxiliary polypeptide, designated the beta subunit, which plays an important role in their regulation. Despite considerable functional semblences and abilities to interact with structurally similar alpha subunits, however, there is considerable structural diversity among the beta subunits. In this review, we discuss the similarities and differences in the structures and functions of the beta subunits of the voltage-dependent K(+), Ca(2+), and Na(+) channels.

Calcium Channels↗
PubMed2001

Synchrotron radiation circular dichroism spectroscopy: vacuum ultraviolet irradiation does not damage protein integrity.

Synchrotron radiation circular dichroism (SRCD) spectroscopy is an emerging technique for sensitive determination of protein secondary structures and for monitoring of conformational changes. An important issue for its adoption as a useful technique is whether the high-intensity low-wavelength vacuum ultraviolet radiation in the SRCD chemically damages proteins. In this paper, using horse myoglobin as a test sample, it is shown that extensive irradiation in the SRCD does not produce any change in the chemical nature of the protein as detected by either SDS gel electrophoresis or mass spectrometry. In addition, no changes in the protein secondary structure are detectable from the SRCD spectra after extensive exposure to the SRCD beam.

Circular Dichroism↗
PubMed2001

Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics.

Recent developments in instrumentation and bioinformatics show that the technique of synchrotron radiation circular dichroism spectroscopy can provide novel information on protein secondary structures and folding motifs, and has the potential to play an important role in structural genomics studies, both as a means of target selection and as a high-throughput, low-sample-requiring screening method. This is possible because of the additional information content in the low-vacuum ultraviolet wavelength data obtainable with intense synchrotron radiation light sources, compared with that present in spectra from conventional lab-based circular dichroism instruments.

Circular Dichroism↗
PubMed2001

Peptaibols: models for ion channels.

Peptaibols are membrane-active polypeptides isolated from fungal sources. They are characterized by the presence of an unusual amino acid, alpha-aminoisobutyric acid, and a C-terminal hydroxylated amino acid. Peptaibols exhibit antibiotic activity against bacteria and fungi. Their amphipathic nature allows them to self-associate into oligomeric ion-channel assemblies which span the width of lipid bilayer membranes. Over 200 peptaibol sequences have been reported to date, which are compiled in the Peptaibol Database at http://www.cryst.bbk.ac.uk/peptaibol. Alignments of these sequences have been carried out in order to define a series of related subfamilies (SFs) with common sequence features thought to be important for channel formation. Crystal structures determined for a number of peptaibols from the various SFs provide the bases both for modelling of the channel structures and for modelling structures of other members of the same SFs.

Alamethicin↗
PubMed2000

In vitro membrane-inserted conformation of the cytochrome b(5) tail.

The cytochrome b(5) tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b(5) to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b(5) have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment.

Amino Acid Sequence↗
PubMed2000

Transmembrane peptide NB of influenza B: a simulation, structure, and conductance study.

The putative transmembrane segment of the ion channel forming peptide NB from influenza B was synthesized by standard solid-phase peptide synthesis. Insertion into the planar lipid bilayer revealed ion channel activity with conductance levels of 20, 61, 107, and 142 pS in a 0.5 M KCl buffer solution. In addition, levels at -100 mV show conductances of 251 and 413 pS. A linear current-voltage relation reveals a voltage-independent channel formation. In methanol and in vesicles the peptide appears to adopt an alpha-helical-like structure. Computational models of alpha-helix bundles using N = 4, 5, and 6 NB peptides per bundle revealed water-filled pores after 1 ns of MD simulation in a solvated lipid bilayer. Calculated conductance values [using HOLE (Smart et al. (1997) Biophys. J. 72, 1109-1126)] of ca. 20, 60, and 90 pS, respectively, suggested that the multiple conductance levels seen experimentally must correspond to different degrees of oligomerization of the peptide to form channels.

Amino Acid Sequence↗
PubMed2000

Synchrotron radiation circular-dichroism spectroscopy as a tool for investigating protein structures.

This paper reviews the use of synchrotron radiation circular dichroism (SRCD) spectroscopy for examining protein structures, discussing the differences between conventional circular dichroism and SRCD, as well as examples of what SRCD studies have revealed about protein structures to date. It further discusses the future potential of the technique, including roles in structural genomics, membrane protein structure elucidation, relationships to crystallographic studies, and protein folding and dynamics.

Journal Article↗
PubMed2000

Common structural features in gramicidin and other ion channels.

This review compares and contrasts the structures of several different types of ion channels with known three-dimensional structures, including gramicidin and the family of peptaibol channels, as well as the Streptomyces lividans potassium channel, to reveal common features in their structures that relate to their functional roles in ion binding and transport across membranes. Specifically, the locations of aromatic amino acids, the dimensions of the molecules, the multimeric nature of the channels and the roles of hydrogen bonds in stabilising such structures, the means by which the channels open and close, and the chemical nature of the groups which make up the channel lumen are discussed. The emphasis is on the commonality of features found in model channels, which may ultimately be found in other biological channel structures.

Amino Acid Sequence↗
PubMed2000

Systematic review of randomized trials of the effect of exercise on bone mass in pre- and postmenopausal women.

Studies of the effect of exercise programs on bone mass appear inconsistent. Our objective was to systematically review and meta-analyze randomized trials of the effect of exercise on bone mass in pre- and postmenopausal women. A computerized MEDLINE search was conducted for the years 1966-1997. Thirty-five randomized trials were identified. Meta-analytic methods were used to statistically pool results of studies of the effect of impact (e.g., aerobics) and non-impact (e.g., weight training) exercise on the lumbar spine and femoral neck. The most studied bone site was the lumbar spine in postmenopausal women (15 studies), where both impact [1.6% bone loss prevented, 95% confidence intervals (CI): 1.0%-2.2%] and non-impact (1.0%, 95% CI: 0.4%-1.6%) exercise programs had a positive effect. Results for the lumbar spine in premenopausal women (eight studies) were similar: 1.5% (95% CI: 0.6%-2.4%) less bone loss (or net gain) after impact exercise and 1.2% (95% CI: 0.7%-1.7%) after non-impact exercise. Impact exercise programs appeared to have a positive effect at the femoral neck in postmenopausal women (five studies), 1.0% (95% CI: 0.4%-1.6%) bone loss prevented, and possibly in premenopausal women, 0.9% (95% CI: -0.2%-2.0%) bone loss prevented. There were too few trials to draw conclusions from meta-analyses of the effect of non-impact exercise on the neck of femur. This systematic review of randomized trials shows that both impact and non-impact exercise have a positive effect at the lumbar spine in pre- and postmenopausal women. Impact exercise probably has a positive effect at the femoral neck. More studies are required to determine the optimal intensity and type of exercise.

Animals↗