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B A Wallace

Publications and source records attributed to B A Wallace.

At least 73 records · Page 4Linked to original sources
PubMed1997

Slow alpha helix formation during folding of a membrane protein.

Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer.

Bacteriorhodopsins↗
PubMed1997

Solvent effects on the conformation and far UV CD spectra of gramicidin.

Solvent effects on the far-uv CD spectra of the polypeptide gramicidin have been studied systematically in a series of alcohols of increasing chain length, ranging from methanol to dodecanol. The effects observed are of two types: primary, involving a change in the equilibrium mixture of conformers present, and secondary, involving a shift in the spectral peak positions as a function of solvent polarizability. To quantitate the primary effect, the ratio of the individual conformers present was estimated by deconvolution of the spectra into their component species. For short chain length alcohols, both parallel and antiparallel double helices are found in considerable abundance. As the solvent chain length is increased and its polarity is decreased, the left-handed antiparallel double helical species is favored. For all alcohols with chain lengths of four or more carbon atoms, the ratio of the conformers present remains relatively constant. To quantitatively examine the secondary effect, the magnitudes of the spectral shifts on the dominant conformer (species 3) have been correlated with the dielectric constants and refractive indices of the solvents, thereby indicating what underlying physical properties are responsible for these shifts. This work thus demonstrates that for gramicidin, a flexible polypeptide, the solvent effects on the CD spectra can be resolved into two types: changes due to the mixture of conformers present and shifts in the spectral characteristics. Both effects need to be considered when interpreting CD spectra in terms of secondary structure for this and other polypeptides in nonaqueous solutions.

Alcohols↗
PubMed1997

The effects of calcium ions on double helical forms of gramicidin.

The effects of binding calcium ions to the double helical forms of gramicidin present in methanol solution were examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. It was found that calcium ions principally alter the relative composition of the equilibrium mixture of gramicidin conformers present in the solvent. In the absence of calcium, both parallel and antiparallel double helices are present. However, the addition of small amounts of Ca2+ shifts the equilibrium towards the left-handed parallel double helical form. This conformational change prevents monovalent cations (caesiums) from binding to the gramicidin double helix, and even converts the shorter, wider anti-parallel double helical form normally produced in the presence of caesium into the longer, narrower parallel double helical form. Furthermore, a temperature study showed that calcium ions tend to stabilize this form relative to the ion-free forms. The conformation of gramicidin is further changed, becoming a disordered structure, when the concentration of Ca2+ is raised. Thus, the binding of divalent calcium ions has a number of dramatic effects on the conformations of gramicidin present in solution.

Calcium↗
PubMed1997

Structure of the CLN3 gene and predicted structure, location and function of CLN3 protein.

The genomic sequence of the human CLN3 gene, which is defective in juvenile onset neuronal ceroid lipofuscinosis (Batten disease) is being delineated using a variety of methods. A Saccharomyces cerevisiae gene, YHC3 (for Yeast Homologue to human CLN3), which is highly similar to the human disease gene, has been identified by computer-aided homology searching. Topology predictions indicate the CLN3 protein contains six transmembrane segments. Most similarity between the human and yeast proteins lies either in the transmembrane segments or along one face of the predicted protein structure.

Adolescent↗
PubMed1997

Circular dichroism studies of human big-endothelin-1 (Big ET-1).

The circular dischroism (CD) spectra of human endothelin (ET-1) and its precursor (Big ET-1) have been compared in order to provide information on the secondary structure of Big ET-1. It appears that the secondary structures of the common parts of the two molecules are very similar and that the additional C-terminal residues in Big ET-1 may contain both helical and sheet components, information which may be of use in modeling studies of the Big ET-1 structure. In studies of the pH dependence of the conformation of Big ET-1, it was found that Big ET-1 adopts similar structures at pH 6.0 and 7.0, but has a subtly different conformation at pH 8.0 that may result in a reduced susceptibility to proteolysis at this pH. This difference may be in the conformation of a turn-type structure, producing a less accessible peptide bond in the molecule at the site of cleavage.

Circular Dichroism↗
PubMed1996

Solution structure of a parallel left-handed double-helical gramicidin-A determined by 2D 1H NMR.

The structure of a parallel left-handed double-helical form of gramicidin was detected by circular dichroism spectroscopy and determined using 500 and 600 MHz NMR in CaCl2/methanol solution. Measurements of TOCSY, DQF-COSY and NOESY spectra were converted into 604 distance and 48 torsional angle constraints for structure calculations. Stereospecific assignments and chi 1 angles were calculated using 3J alpha beta, d alpha beta (i,i), dN beta(i,i) and dN gamma(i,i). chi 2 angles were determined using d alpha beta(i,i), dN beta(i,i), d beta delta(i,i), dN gamma(i,i) and d alpha gamma(i,i). The calculations of initial structures were performed using the distance geometry/simulated annealing method in XPLOR. The initial structures were further refined and energy minimized using simulated annealing/molecular dynamics methods. Back-calculations for every generated structure were also performed to check their consistency with the experimental data. 187 final structures with no violations above the threshold conditions (0.05 A, 5 degrees, 5 degrees, 0.5 A and 5 degrees for bonds, angles, improper, NOE and cdihe, respectively) were produced from the 200 initial structures. Twenty structures with the lowest NOE energies were used for further analysis. The average r.m.s. deviations for the 20 structures are 0.64 A for backbone and 1.1 A for all non-hydrogen atoms. Gramicidin in this form, with approximately 5.7 residues per turn, is a parallel double helical dimer. The length along the helix axis is about 30 A and the inner pore diameter varies from 1 to 2 A. It is different from all other gramicidin structures determined to date. The presence of Ca2+ stabilises a conformation that prevents the binding of monovalent cations. It is likely that this structure is related to a non-channel, antibiotic role of gramicidin.

Amino Acid Sequence↗
PubMed1996

A model for Batten disease protein CLN3: functional implications from homology and mutations.

In an attempt to understand the molecular nature of Batten disease, we have examined the amino acid sequence of the affected CLN3 gene product (The International Batten Disease Consortium (1995) Cell 82, 949-957) and the site-specific mutations which give rise to the biological defect. Homology searches and molecular modeling have led to the development of a model for the folding and disposition of the protein, possibly within a mitochondrial membrane. High homology with a yeast protein of unknown function suggests a strong evolutionary conservation of function. We speculate that a possible role for the protein may be in chaperoning the folding/unfolding or assembly/ disassembly of other proteins, specifically subunit c of the mitochondrial ATP synthase complex.

Amino Acid Sequence↗
PubMed1996

A molecular model for human Big-Endothelin-1 (Big ET-1).

A molecular model has been developed for human Big Endothelin-1, which is the immediate precursor to the potent vasoconstrictor polypeptide endothelin-1 and the target of the highly specific endothelin converting enzyme. This model is produced by a threading algorithm protocol and is consistent with all the currently available structural and biochemical data for this molecule.

Algorithms↗
PubMed1996

Binding of alkaline cations to the double-helical form of gramicidin.

Gramicidin is a polypeptide antibiotic that forms monovalent cation-specific channels in membrane environments. In organic solvents and in lipids containing unsaturated fatty acid chains, it forms a double-helical "pore" structure, in which two monomers are intertwined. This form of gramicidin can bind two cations inside its lumen, and the crystal structures of both an ion complex and an ion-free form have been determined. In this study, we have used circular dichroism (CD) spectroscopy to examine the binding mechanism and the binding constants (K1 and K2) of cations to gramicidin in the double helical form in methanol solution. The dramatic change in optical rotation in the far-ultraviolet CD spectrum of gramicidin provides a useful tool for monitoring the binding. The binding mechanism appears to involve a large conformation change associated with the binding of ions to the first of the two sites. The calculated values for the K1 binding constants for alkaline cations are considerably smaller than the K2 binding constants. The order of binding affinity for alkaline cations is similar to that for the helical dimer "channel" form of gramicidin, i.e., Cs+ approximately Rb+ > > K+ > Li+, but in comparison to the helical dimer form, the binding to double-helical dimers is dominated by a cation size-dependent conformational change in the gramicidin structure.

Amino Acid Sequence↗
PubMed1996

HOLE: a program for the analysis of the pore dimensions of ion channel structural models.

A method (HOLE) that allows the analysis of the dimensions of the pore running through a structural model of an ion channel is presented. The algorithm uses a Monte Carlo simulated annealing procedure to find the best route for a sphere with variable radius to squeeze through the channel. Results can be displayed in a graphical fashion or visualized with most common molecular graphical packages. Advances include a method to analyze the anisotropy within a pore. The method can also be used to predict the conductance of channels using a simple empirically corrected ohmic model. As an example the program is applied to the cholera toxin B-subunit pentamer. The compatibility of the crystal structure and conductance data is established.

Algorithms↗
PubMed1995

Comparison of the structures of the endothelin A receptor antagonists BQ123 and N-methyl leucine BQ123 with the crystal structure of the C-terminal tail of endothelin-1.

The functionally important regions of the cyclic pentapeptide endothelin A receptor antagonist BQ123 are shown to correlate with the structure of the C-terminal tail of endothelin-1, as found in the recently-determined X-ray crystal structure. Residues 18 and 21 of endothelin-1 are spatially juxtaposed such that they superpose extremely well with D-Asp and D-Trp of the antagonist, consistent with the residues on this surface of the endothelin helix being important for binding. This study provides new information on the three-dimensional nature of the endothelin A receptor binding site which may prove useful for rational drug design.

Amino Acid Sequence↗
PubMed1995

Effects of local environment on the circular dichroism spectra of polypeptides.

The secondary structures of proteins may be estimated by analysis of their circular dichroism spectra using techniques which empirically fit component spectra derived from water-soluble protein databases. However, local environment effects red- or blue-shift peptide energy transitions primarily through solvent polarization effects and may be an especially important factor for proteins in nonaqueous environments. In this study the reference basis spectra were deconvoluted into three component Gaussian curves corresponding to each of the peptide absorbances in the wavelength range from 190 to 240 nm. These Gaussians were then systematically shifted and utilized in the analyses of the spectra of crambin solubilized in a variety of nonaqueous solvents, including that of the polypeptide associated with small unilamellar phospholipid vesicles. Crambin was selected since its well-characterized structure was found to retain native structure in a variety of nonaqueous environments. This method was more successful in estimating the helical content of this protein than conventional methodologies. This type of analysis may prove to be appropriate for estimating the secondary structure of proteins found in nonaqueous environments, such as membrane proteins.

Alcohols↗
PubMed1995

A comparison of X-ray and NMR structures for human endothelin-1.

Direct comparisons between the recently solved X-ray and NMR structures of human endothelin-1 with respect to secondary structure, RMS deviations, surface accessibilities, and side-chain conformers indicate important differences in conformation, especially in the C-terminus, but also in the central loop region, that are important for defining the specificity of binding. These differences are larger than seen for other X-ray and NMR structures that have been compared. Comparisons between the X-ray structure and the NMR NOE constraints highlight the regions of flexibility and environment-induced diversity in the endothelin structures.

Computer Graphics↗
PubMed1995

The crystal structure of human endothelin-1 and how it relates to receptor binding.

Several different three-dimensional structures have recently been proposed for human endothelin-1 (ET-1) based on x-ray crystallographic, NMR spectroscopic, and modeling studies. All differ considerably in the regions that are critical for receptor binding, i.e., the central loop and the C-terminal tail, which is helical in the crystal structure but not helical in any of the other structures. In this study we examined the various ET structures in light of binding and vasoactivity data available for naturally occurring isoforms and synthetic mutants of ET. These studies strongly support the crystal structure alone as being biologically relevant with respect to receptor binding. Furthermore, molecular modeling studies based on the crystal structure of ET-1 have produced models for the ET-2 and ET-3 isoforms that suggest geometric properties for the receptor selectivity pocket of the ET(A) receptor. From these studies, it appears that the crystal structure may prove useful for aiding our understanding of the nature of the receptor/ligand binding sites and for providing a basis for the design of new agonists and antagonists.

Binding Sites↗
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

The crystal structure of human endothelin.

The three-dimensional structure of the vasoactive polypeptide endothelin, the most potent vasoconstrictor yet identified, has been determined by X-ray crystallography to 2.18 A resolution. This intermediate-sized structure was solved by molecular replacement techniques using a fragment of an NMR-derived model for initial phasing of the data. However, comparisons of the final X-ray structure with the many diverse models derived from NMR data indicate some important differences, especially in the carboxy-terminal region of the molecule: the entire carboxy terminal tail (residues 16-21) is helical in the crystal structure, but not in any of the NMR structures. This may be a functionally significant difference as this region is crucial for receptor binding and vasoactivity.

Amino Acid Sequence↗