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B A Wallace

Publications and source records attributed to B A Wallace.

At least 55 records · Page 3Linked to original sources
PubMed2000

Modeling and docking the endothelin G-protein-coupled receptor.

A model of the endothelin G-protein-coupled receptor (ET(A)) has been constructed using a segmented approach. The model was produced using a bovine rhodopsin model as a template for the seven transmembrane alpha-helices. The three cytoplasmic loop regions and the C-terminal region were modeled on NMR structures of corresponding segments from bovine rhodopsin. The three extracellular loops were modeled on homologous loop regions in other proteins of known structure. The N-terminal region was modeled as a three-helix domain based on its homology with a hydrolase protein. To test the model, the FTDOCK algorithm was used to predict the ligand-binding site for the crystal structure of human endothelin. The site of docking is consistent with mutational and biochemical data. The principal sites of interaction in the endothelin ligand all lie on one face of a helix that has been implicated by structure-activity relationship studies as being essential for binding. As further support for the model, attempts to dock bigET, an inactive precursor to endothelin that does not bind to the receptor, found no sites for tight binding. The model of the receptor-ligand complex produced forms a basis for rational drug design of agonists and antagonists for this G-protein-coupled receptor.

Algorithms↗
PubMed2000

Expression and characterization of the human endothelin-A-receptor in Pichia pastoris: influence of N-terminal epitope tags.

Knowledge of the three-dimensional structure of the endothelin-A receptor (ET(A)) will provide important information for rational drug design of antagonists and agonists. In order to produce correctly folded and active receptor protein for physical characterization by such techniques as X-ray crystallography and circular dichroism spectroscopy, we have cloned and expressed the human ET(A)-receptor in Pichia pastoris. The expression constructs all contained signal sequences to direct the expressed protein to the membrane fraction. Fidelity of folding and the subtype specificity of the expressed receptor was demonstrated by ligand binding studies using 125I-labelled endothelin-1 (ET-1). Expression of receptor with two different N-terminal epitope 'tags' had little effect on the affinity of ET-1 for the receptor. The strain of P. pastoris employed did influence the quantity of receptor expressed.

Humans↗
PubMed1999

Modelling of a voltage-dependent Ca2+ channel beta subunit as a basis for understanding its functional properties.

Structure prediction methods have been used to establish a domain structure for the voltage-dependent calcium channel beta subunit, beta1b. One domain was identified from homology searches as an SH3 domain, whilst another was shown, using threading algorithms, to be similar to yeast guanylate kinase. This domain structure suggested relatedness to the membrane-associated guanylate kinase protein family, and that the N-terminal domain of the beta subunit might be similar to a PDZ domain. Three-dimensional model structures have been constructed for these three domains. The extents of the domains are consistent with functional properties and mutational assays of the subunit, and provide a basis for understanding its modulatory function.

Amino Acid Sequence↗
PubMed1999

Do the structures of big ET-1 and big ET-3 adopt a similar overall fold? Consequences for endothelin converting enzyme specificity.

Big ET-1 and big ET-3 are precursor peptides which render endothelin-1 (ET-1) and endothelin-3 (ET-3) relatively unreactive and resistant to proteolytic cleavage. Big ET-1 is cleaved in vivo by ECE-1 (endothelin-converting enzyme), and big ET-3 is also cleaved but apparently to a significantly lesser extent by this enzyme. To shed light on the relation between structure and function, circular dichroism (CD) spectroscopy and homology modeling were used to determine whether big ET-1 and big ET-3 adopt similar secondary and tertiary structures. Analyses of the CD spectra and thermal denaturation indicate they have similar secondary structures and thermal stabilities. Superposition of the modeled coordinates of both peptides indicates that they can adopt the same overall fold except in the C-terminal residues, 34-38 in big ET-1 and 34-41 in big ET-3. This region corresponds to an area of complete sequence heterogeneity between the two peptides. A model has been developed which has a loop for residues 27-30 (HVVP in big ET-1), which have previously been demonstrated to be essential for eliciting efficient hydrolysis of the W21-V22 bond in big ET-1 and which have the sequence QTVP in big ET-3. Differences in affinity between big ET-1 and big ET-3 for ECE-1 thus appear to be due solely to sequence variations in the local region of the cleavage site.

Amino Acid Sequence↗
PubMed1999

Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein.

A random library of phage displayed peptides was screened for binding to a biotinylated derivative of paclitaxel (Taxol). Affinity-selected peptides were analyzed for similarity to human proteins. There was no significant similarity between the paclitaxel-selected peptides and tubulin. However, a subset of the peptides was identified that exhibits significant similarity to a non-conserved region of the anti-apoptotic human protein Bcl-2: ELISA assays confirmed binding of paclitaxel to Bcl-2, and circular dichroism spectroscopy demonstrated that a substantial conformational change accompanies this binding. In vivo, treatment with paclitaxel has been shown to lead to Bcl-2 inactivation with concomitant phosphorylation of residues in a disordered, regulatory loop region of the protein. Similarity between paclitaxel-selected peptides and this loop region implicate these residues in drug binding, and suggest that the apoptotic action of paclitaxel may involve the binding of paclitaxel to Bcl-2. These results demonstrate that peptides displayed on the surface of bacteriophage particles can mimic the ligand-binding properties of disordered regions of proteins.

Amino Acid Sequence↗
PubMed1999

Tryptophans in membrane proteins. X-ray crystallographic analyses.

While tryptophans are generally found in low abundance in soluble proteins, in many integral membrane proteins they comprise a significantly higher proportion of the amino acid composition. Now that crystal structures are available for a number of membrane proteins, it has been possible to examine the distribution and disposition of the tryptophans within these structures. The tryptophan locations with respect to the lipid bilayer (along the direction normal to the membrane surface) are strikingly non-uniform in nearly all of the membrane proteins examined. They tend to cluster at the interface between the polar head group region and the hydrophobic interior, in a relatively uniform layer just below the surface. In many cases, their distributions with respect to the extra- and intra-cellular surfaces tend to be asymmetric. These observations provide evidence for possible structural roles for tryptophans in transmembrane sheets and helices, where they may play a part in the stabilization of the transmembrane segments and perhaps in the orientation and bilayer insertion processes.

Bacteria↗
PubMed1999

The molecular-replacement solution of an intermediate-sized helical polypeptide, antiamoebin I.

The successful use of molecular-replacement methods for the solution of the intermediate-sized helical polypeptide antiamoebin I required the careful consideration of a number of parameters and exhibited some unusual characteristics when compared with molecular-replacement solutions of globular proteins. High-resolution data were required owing to several features, including the comma-like shape of the molecule (which results in a pseudo-symmetric structure at low resolution), the relative uniformity of the structure in the direction along the helix axis and the small differences between the two independent molecules in the P1 asymmetric unit. Other parameters which were important for the solution of this relatively low solvent content closely packed cell included the radius of integration, the use of normalized structure factors and especially the choice of starting model.

Amino Acid Sequence↗
PubMed1999

X-ray crystallographic structures of gramicidin and their relation to the Streptomyces lividans potassium channel structure.

Gramicidin has been used extensively as a model system for structure/function studies of ion channels. Long before crystals of other ion channel proteins were produced, crystals of gramicidin had been prepared, even though it was many years before the first forms of those crystals were solved. There now exist a large number of crystal structures of both uncomplexed and ion-complexed forms of gramicidin crystallized from organic solvents. In all these crystals, the molecules are double helices, although they differ in helical pitch, handedness and side chain orientations, depending on the conditions used for crystallization. Since many of these structures have been discussed in detail in a recent review (Wallace 1998), this chapter concentrates on recently reported structures and how they relate to previously described X-ray and NMR structures. It also discusses how the crystal structure of a K+ complex of gramicidin relates to the recently solved structure of a K+ complex of the potassium channel from Streptomyces lividans and argues that this demonstrates that gramicidin is indeed a good model structure for biological ion channels, despite the presence of D-amino acids in its sequence.

Anti-Bacterial Agents↗
PubMed1998

Antiamoebin can function as a carrier or as a pore-forming peptaibol.

Antiamoebin is a 16-residue polypeptide whose crystal structure and lytic activity in membrane vesicles have recently been reported. It is a bent helical molecule and a member of the peptaibol family of antibiotics. Under conditions which produce voltage-dependent conductance activity by other members of the family, no single-channel conductance was detected for antiamoebin, and a carrier-like mechanism was put forward to account for its mode of action. We now present evidence for pore formation that is largely voltage-insensitive, with large amplitude single-channel events on top of a background conductance that may account for the previously proposed carrier-like activity. Thus, antiamoebin may be the first instance of a peptide which can function both as an ion carrier and a pore former.

Amino Acid Sequence↗
PubMed1998

The structure and function of antiamoebin I, a proline-rich membrane-active polypeptide.

BACKGROUND: Antiamoebin is a member of the peptaibol family of polypeptides and has a unique antibiotic activity: it acts as an antiamoebic agent, but does not effectively haemolyze erythrocytes even though it does exhibit membrane-modifying activity. RESULTS: The structure of antiamoebin I has been determined by X-ray crystallography at 1.4 A resolution. The molecule forms a helical structure, which, as a result of the presence of a number of proline and hydroxyproline residues, has a deep bend in the middle. Circular dichroism spectroscopy, single-channel conductance studies and fluorescence diffusion studies suggest a mode of ion transport that is entirely different from that of the other two members of the peptaibol family (alamethicin and zervamicin) whose structures and functions have been examined in detail. CONCLUSIONS: The structure of the polypeptide has been determined and a functional model for its mode of action in membranes is presented. Although under some conditions antiamoebin may form ion channels, unlike the closely related alamethicin and zervamicin polypeptides, its major membrane-modifying activity appears to be as an ion carrier.

Alamethicin↗
PubMed1998

Shifting the equilibrium mixture of gramicidin double helices toward a single conformation with multivalent cationic salts.

The conformation of the polypeptide antibiotic gramicidin is greatly influenced by its environment. In methanol, it exists as an equilibrium mixture of four interwound double-helical conformers that differ in their handedness, chain orientation, and alignment. Upon the addition of multivalent cationic salts, there is a shift in the equilibrium to a single conformer, which was monitored in this study by circular dichroism spectroscopy. With increasing concentrations of multivalent cations, both the magnitude of the entire spectrum and the ratio of the 229-nm to the 210-nm peak were increased. The spectral change is not related to the charge on the cation, but appears to be related to the cationic radius, with the maximum change in ellipticity occurring for cations with a radius of approximately 1 A. The effect requires the presence of an anion whose radius is greater than that of a fluoride ion, but is otherwise not a function of anion type. It is postulated that multivalent cations interact with a binding site in one of the conformers, known as species 1 (a left-handed, parallel, no stagger double helix), stabilizing a modified form of this type of structure.

Amino Acid Sequence↗
PubMed1998

Phospholipid chain length alters the equilibrium between pore and channel forms of gramicidin.

Gramicidin is an excellent model system for studying the passage of ions through biological membranes. The conformation of gramicidin is well defined in many different solvent and lipid systems, as are its conductance and spectroscopic properties. It is a polymorphic molecule that can adopt two different types of structure, the double helical "pore" and the helical dimer "channel". This study investigated the influence of the acyl chain length of membrane phospholipids on the conformations adopted by gramicidin. We used circular dichroism spectroscopy to examine the conformational equilibrium between the pore and channel forms in small unilamellar vesicles of phosphatidylcholine with acyl chain lengths of 18, 20 and 22 carbons. Our results show that in C18 and C20 lipids almost all the gramicidin is in the channel form, while in the longer C22 lipids the equilibrium shifts in favour of pore conformations, such that they form up to 43% of the total population. This change is attributed to the ability of the double helical conformation to tolerate more hydrophobic mismatch than the helical dimer, perhaps due to the greater number of stabilising intermolecular hydrogen bonds.

Cell Membrane↗
PubMed1997

Secondary solvent effects on the circular dichroism spectra of polypeptides in non-aqueous environments: influence of polarisation effects on the far ultraviolet spectra of alamethicin.

Secondary solvent effects on the far ultraviolet circular dichroism spectra of the polypeptide alamethicin have been studied systematically in a series of alcohols. The magnitudes of the shifts have been correlated with the physical properties of the solvents in an attempt to discover the underlying physical principles responsible for these shifts. The solvent effect in non-aqueous solvents generally produces spectral transitions with peaks found at longer wavelengths than those in aqueous solution, and is correlated with increasing refractive indices and with decreasing dielectric constants of the solvents. It appears that polarisation effects are the major contributors to the interactions between the chromophore and solvent molecules, and hence give rise to the red shift. It is clear that this secondary solvent effect is an important factor which should be considered in the examination and estimation of polypeptide secondary structures in non-aqueous solvents and membranes.

Journal Article↗
PubMed1997

Crystal structure of the gramicidin/potassium thiocyanate complex.

The hydrophobic channel-forming polypeptide gramicidin adopts a left-handed antiparallel double helix conformation with 6.4 residues per turn when in complex with monovalent cation salts in a methanol environment. The crystal structure of the gramicidin/potassium thiocyanate complex (a = 32.06 A, b = 51.80 A, and c = 31.04 A; space group P2(1)2(1)2(1)) has been solved to 2.5 A with an R-factor of 0.193. In the structure, binding sites for the cations are formed by the polypeptide backbone carbonyl groups tilting away from the helix axis toward the ions located in the central lumen. The polypeptide backbone conformations and the side-chain orientations in this potassium complex are significantly different from those in the previously solved gramicidin/caesium chloride crystal complex, due to the requirements for interactions with the smaller sized potassium cation. The locations and numbers of potassium binding sites also differ considerably from the locations and numbers of caesium binding sites in the other structure. Combining information from all the cation binding sites in the two gramicidin/ion complexes produces different views of the three-dimensional structures of a cation as it is transported along a transmembrane pore, and provides an experimental structural basis for modeling the dynamics of peptide-ion binding and ion transport.

Anions↗