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Biomedical subjects

B A Wallace

Publications and source records attributed to B A Wallace.

At least 91 records · Page 5Linked to original sources
PubMed1993

Preliminary crystallization and X-ray analysis of orthorhombic human endothelin.

Human endothelin (ET-1) is a highly potent, ubiquitous, endogenous 21 amino acid residue polypeptide, which acts as a vaso- and bronchoconstrictor. Crystals of the active, uncomplexed form of ET-1 have been grown from aqueous solutions. These crystals, which are long hexagonal prisms, diffract to 2.98 A and are apparently of the space group P222(1) with cell dimensions a = 33.5 A, b = 57.9 A, c = 59.6 A. The six molecules in the asymmetric unit are related by non-crystallographic symmetry operations, which result in a pseudo 6(1) appearance to the data.

Crystallography, X-Ray↗
PubMed1993

Analyses of ligand binding in five endothiapepsin crystal complexes and their use in the design and evaluation of novel renin inhibitors.

Five renin inhibitors were cocrystallized with endothiapepsin, a fungal enzyme homologous to renin. Crystal structures of inhibitor-bound complexes have provided invaluable insight regarding the three-dimensional structure of the aspartic proteinase family of enzymes, as well as the steric and polar interactions that occur between the proteins and the bound ligands. Beyond this, subtleties of binding have been revealed, including multiple subsite binding modes and subsite interdependencies. This information has been applied in the design of novel potent renin inhibitors and in the understanding of structure-activity relationships and enzyme selectivities.

Amino Acid Sequence↗
PubMed1993

Temperature dependence of the interaction of alamethicin helices in membranes.

The interaction of the voltage-dependent channel-forming peptide alamethicin with dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) has been studied using circular dichroism spectroscopy over a range of wavelengths and temperatures. Evidence is presented for the existence of two distinct membrane-bound states of the peptide which reflect different extents of peptide-peptide interaction. An elevated temperature is found to diminish the apparent peptide-peptide interaction. These results provide insight into the general problem of helix-helix interaction in membranes and provide experimental support for the proposal [Popot, J. L., & Engelman, D. M. (1990) Biochemistry 29, 4031-4037] that these interactions can be enthalpically favorable.

Alamethicin↗
PubMed1993

Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra.

The secondary structure of bacteriorhodopsin is known from electron-diffraction studies, making bacteriorhodopsin a useful test system for analysing environmental influences on membrane proteins using c.d. spectroscopy. The conformational effects of detergent solubilization and incorporation into vesicles of various types were determined by comparison of the calculated secondary structures derived from c.d. spectra with the structure determined from diffraction studies. In addition, two modified forms of the native purple membrane, a shrunken form of the hexagonal lattice and an orthorhombic lattice form, were used to determine the effects of varying membrane fragment size and protein concentration within the membranes. The two different vesicle incorporation procedures yielded bacteriorhodopsin spectra which were nearly identical with each other and very close to the structure calculated from electron-diffraction studies. Solubilization of the native protein in the non-ionic detergent n-octyl glucoside, without subsequent vesicle incorporation, resulted in a significantly altered protein conformation. Organizing the protein in different membrane lattices produced even more apparent deviations from the secondary structure determined by diffraction studies, as a consequence of optical effects caused by the high protein concentrations in the lattices. These studies show the importance of maintaining a 'native' environment, and the influence of particle geometry in interpreting c.d. studies of membrane proteins.

Bacteriorhodopsins↗
PubMed1993

The pore dimensions of gramicidin A.

The ion channel forming peptide gramicidin A adopts a number of distinct conformations in different environments. We have developed a new method to analyze and display the pore dimensions of ion channels. The procedure is applied to two x-ray crystal structures of gramicidin that adopt distinct antiparallel double helical dimer conformations and a nuclear magnetic resonance (NMR) structure for the beta6.3 NH2-terminal to NH2-terminal dimer. The results are discussed with reference to ion conductance properties and dependence of pore dimensions on the environment.

Amino Acid Sequence↗
PubMed1993

An analysis of 9,918 consecutive perioperative autotransfusions.

The efficacy of perioperative autotransfusion (PAT) can be evaluated by the reduction in homologous transfusion accompanying its use. An alternative approach is to evaluate the amount of blood salvaged and retransfused. An analysis of 9,918 consecutive PAT procedures in various surgical specialties revealed that the average return of autologous blood salvaged was equivalent to 2.61 units of erythrocytes ("packed cells"). Cardiac operation had the greatest average number of units recovered (4.65), while orthopedic operation had the least (1.05). This method of analysis demonstrates that significant quantities of blood can be salvaged during PAT procedures.

Blood Loss, Surgical↗
PubMed1992

Effects of pH on conformational properties related to the toxicity of Bacillus thuringiensis delta-endotoxin.

The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects. Upon dissolution at pH > 9 it yields a protein subunit called protoxin. Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect. It is known that this toxic activity decreases considerably above pH 10. In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties. At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx. 26% alpha-helix and approx. 45% beta-structure. As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased. The net secondary structure did not change significantly at pH values below 10. Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values. The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins.

Bacillus thuringiensis↗
PubMed1992

Model ion channels: gramicidin and alamethicin.

We have discussed in some detail a variety of experimental studies which were designed to elucidate the conformational and dynamic properties of gramicidin and alamethicin. Although the behavior of these peptides is by no means fully characterized, these studies have already permitted aspects of ion channel activity to be understood in molecular terms. Studies with gramicidin in a variety of organic solutions have revealed conformational heterogeneity of this peptide; at least five major isomers exist, several of which have been characterized in detail using NMR spectroscopy and X-ray crystallography. When added to lipid membranes gramicidin undergoes a further conformational conversion. Although the conformation of gramicidin in membranes is not as well characterized as the solution conformation(s) and an X-ray structure is not yet available, detailed data, particularly from solid-state NMR studies, continue to become available and a right-handed beta 6.3 helical conformation of the peptide backbone is now generally accepted. Two of these beta 6.3 helices joined at their N-termini are believed to form the conducting channel. The conformational behavior of the side-chains of gramicidin in the membrane-bound form is not well established and several NMR, CD, fluorescence and theoretical studies are now focussed on this. Although the side-chains do not directly contact the permeating ions, they can have distinct effects on conductance and selectivity by altering the electrostatic environment sensed by the ion. The dynamics of both side-chain and backbone conformations of gramicidin appear critical to a detailed understanding of the ion transport process in this channel. As the description of the membrane-bound conformation of gramicidin becomes more detailed, simulations of ion transport using computational methods are likely to improve and will further our understanding of the processes of ion transport. As well as internal motion of the backbone and side-chains, gramicidin undergoes rotational and translational motion in the plane of the membrane. These motions do not appear to be essential for the process of ion transport but can affect channel lifetime since lifetime is determined by the rate of association and dissociation of gramicidin monomers. Gramicidin-membrane interactions are also likely to be involved in the frequency of occurrence of channel subconductance states, the frequency of channel flickering and fundamentally in the stability of the membrane-bound gramicidin conformation. Alamethicin forms channels in membranes which are strongly voltage-dependent. The molecular origin of voltage-dependent conductances has been a fundamental problem in biophysics for many years.(ABSTRACT TRUNCATED AT 400 WORDS)

Alamethicin↗
PubMed1991

Protein-chromophore interactions in bacteriorhodopsin: the effects of a change in surface potential.

The chromophore retinal is bound to bacteriorhodopsin via a protonated Schiff base linkage. The retinal binding site is reported to be buried in the transmembrane portion of the protein, distant from the membrane surfaces. When bound to bacteriorhodopsin, the absorption maximum of retinal is red-shifted from 366 nm to 568 nm producing a purple color. This color persists across a wide pH range. However, when the pH is raised above 12.0, the membranes become pink in color, while at pH values of 3.0 or below, a blue color is produced. The blue color can also be obtained by removing the divalent cations bound to the surface of the protein. In this study, bacteriorhodopsin was examined by circular dichroism and absorption spectroscopy to determine if protein conformational changes were associated with the color shifts. It was found that although the retinal chromophore can be completely removed by bleaching with hydroxylamine with no significant influence on the secondary structure of the protein, a change in the surface charge of bacteriorhodopsin results in measurable conformational change in the protein, which apparently affects the nature of the retinal binding site.

Bacteriorhodopsins↗
PubMed1991

Co-crystals of gramicidin A and phospholipid. A system for studying the structure of a transmembrane channel.

Single crystals of a complex of gramicidin A, a transmembrane channel-forming polypeptide, and dipalmitoyl phosphatidylcholine, a phospholipid, have been prepared and characterized by X-ray diffraction. They belong to space group P222(1), with unit cell dimensions a = 26.8 A, b = 27.5 A, c = 32.8 A. The asymmetric unit appears to be a complex of one gramicidin monomer and two phospholipid molecules. The unit cell dimensions, space group, and chemical composition are compatible with lipids packing in a bilayer-like motif, and an end-to-end association of gramicidin monomers to form a functional dimeric unit. The crystals diffract to 2 A and are suitable for structural studies by single-crystal X-ray analysis. This represents the first example of a well-ordered crystalline channel complexed with lipids, and solution of its structure may give insight into mechanisms of ion transport across membranes.

Gramicidin↗
PubMed1990

The use of single-wavelength anomalous scattering to solve the crystal structure of a gramicidin A/caesium chloride complex.

Single-wavelength Cu K alpha anomalous scattering has been used to determine the structure of a crystalline complex of gramicidin A and caesium chloride. The asymmetric unit in these crystals, with space group P2(1)2(1)2(1) and a = 32.118 (6), b = 52.103 (12), c = 31.174 (7) A, contains four independent monomers (two dimers) of the pentadecapeptide. This structure falls in an intermediate size range for which direct methods and multiple isomorphous replacement are generally not successful for obtaining phase information. However, using the Bijvoet differences and the partial structure of the caesium atoms which have been incorporated in the crystals, it has been possible to obtain information on this crystal form. Because the caesium atoms dominate the scattering of these crystals, inclusion of the Friedel mate information in the restrained least-squares refinement has been essential. These studies extend the utility of single-wavelength anomalous-scattering phase determination to a macromolecular structure in which the partial structure of the anomalous scatterer is large.

Cesium↗
PubMed1990

The secondary structure of gap junctions. Influence of isolation methods and proteolysis.

Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.

Animals↗