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Biomedical subjects

W E Fowler

Publications and source records attributed to W E Fowler.

At least 19 recordsLinked to original sources
PubMed1990

Electron microscopy of human factor V and factor VIII: correlation of morphology with domain structure and localization of factor V activation fragments.

Clotting factor V and factor VIII are each represented by the domain structure A1-A2-B-A3-C1-C2 and share 40% sequence homology in the A and C domains. Rotary-shadowed samples of human factor V and factor VIII were examined in the electron microscope. Single-chain factor V molecules exhibited a globular "head" domain 12-14 nm in diameter. In addition, up to 25% of these molecules showed a rod-like "tail" of up to 50 nm. Glycerol-gradient centrifugation of factor V treated with thrombin partially resolved the factor Va heterodimer from a larger activation peptide of 150 kDa, as determined by gel electrophoresis. Electron microscopy of factor Va revealed globular molecules with several smaller appendicular structures but lacking the tails seen in factor V. Images of the 150-kDa activation peptide showed rod-like structures, similar in width to the tail of intact factor V and approximately 34 nm long. Rotary shadowing was also used to visualize factor VIII that had been fractionated into heterodimers containing heavy chains of distinct sizes. Each factor VIII preparation showed a globular structure approximately 14 nm in diameter, but the associated tails were observed much more frequently with factor VIII heterodimers containing the higher-molecular-weight heavy chains. These results, in conjunction with results of studies using other biophysical techniques, suggest a model in which the A and C domains of each cofactor constitute a globular head and the connecting B domain is contained in a two-stranded tail that is released by thrombin cleavage.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.

Chromatography, Gel↗
PubMed1984

Towards an alignment of the actin molecule within the actin filament.

Electron micrographs of negatively stained actin filament paracrystals and single-layered filament rafts showing different interfilament spacings have been studied and three-dimensional reconstructions have been computed from them. Lateral ordering of the filaments in rafts was lost when interfilament spacings exceeded 8.5 nm, suggesting this distance as an upper limit for the filament diameter. Further, all reconstructions showed the same structural features at the 3 nm resolution level, except that the filaments from ordered single-layered rafts appeared 10-20% wider than those from multi-layered paracrystals. A comparison between electron microscopical and X-ray filament data, and synthetic filaments generated using different tentative molecular models and/or orientations for actin did not allow a single best model to be selected.

Actins↗
PubMed1984

Electron microscopy and image processing applied to the study of protein structure and protein-protein interactions.

We review the application of electron microscopy and image processing at the molecular level to an ever increasing range of biological specimens. Although recent advances have been due in part to development of more sophisticated instrumentation and/or processing algorithms, widespread application of the well-known techniques of image enhancement and structure reconstruction has depended on new strategies of in vitro crystallization and polymerization, some of which are outlined here. We also discuss the use of stoichiometric labeling and/or "cocrystallization" in identifying the different subunits in multisubunit complexes and in studying protein-protein interactions.

Animals↗
PubMed1984

Tubular arrays of the actin-DNase I complex induced by gadolinium.

We describe the preparation and structural analysis of ordered tubular arrays of the actin-DNase I complex. These structures consist of helically stacked rings; each ring is 73 A thick, has a 240 A outer and a 120 A inner diameter, and has 7-fold rotational symmetry. The actin-DNase I complex forms tubes under conditions in which actin alone aggregates into crystalline sheets-i.e., in the presence of the trivalent cation gadolinium. Moreover, upon addition of an equimolar amount of DNase I, crystalline actin sheets are slowly converted to tubes. The rings making the tubes contain a radial dyad axis that may be identical to the dyad axis of the unit cell of the crystalline actin sheet. Evidence is presented for this identification, which in turn allows tentative assignment of actin- and DNase I-containing regions in three-dimensional reconstructions of the rings. The structural analysis presented here may be useful in aligning available three-dimensional molecular models of actin determined from crystals of the actin-DNase I complex and from crystalline actin sheets with each other and ultimately within the biologically important actin filament.

Actins↗
PubMed1983

Structure of the actin molecule determined from electron micrographs of crystalline actin sheets with a tentative alignment of the molecule in the actin filament.

Electron microscopy and image processing of negatively stained crystalline sheets induced from Acanthamoeba actin have been used to yield a three-dimensional reconstruction of the actin molecule, including data to a maximum resolution of 15 A. This model shows actin to be an asymmetric, wedge-shaped molecule. A three-dimensional reconstruction of an averaged, polar actin filament from negatively stained polylysine-induced actin filament paracrystals has also been computed. We show two possible ways in which the wedge-shaped actin molecule from the sheets can be placed into such a filament reconstruction. In both, the major intermolecular contacts are formed on complementary surfaces of the actin subunit and follow the left-handed genetic helix of the filament, a feature also found in the filament reconstruction.

Actins↗
PubMed1983

Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing.

We have compared the appearance and preservation of molecular and supramolecular structures in preparations that were dried in vacuo at room temperature or freeze-dried. Fibrinogen and brain spectrin molecules appear similar in both types of preparation provided that drying at room temperature is performed in the presence of glycerol, which results in an even and reproducible distribution of such molecules (Shotton et al., 1979, J. Mol. Biol. 131, 303-329; Fowler and Erickson, 1979, J. Mol. Biol. 134, 241-249). In the case of crystalline actin sheets, actin filaments, and keratin filaments, freeze-drying preserves structural details that are often completely lost during drying at room temperature, whether or not glycerol is used. On the other hand, keratin filaments prepared by drying in the presence of glycerol display a beaded axial repeat that is probably due to "glycerol decoration." We conclude that although freeze-drying has no clear advantage over glycerol spraying/vacuum-drying in the case of single extended molecules, it may provide insight into the multiple effects of glycerol in specimen preparation. In the case of supramolecular assemblies such as filaments or crystalline sheets, freeze-drying preserves significantly more substructure and surface detail. The loss of such detail during drying at room temperature, probably through collapse phenomena such as distortion and flattening, cannot be prevented by glycerol.

Actins↗
PubMed1983

A consistent picture of the actin filament related to the orientation of the actin molecule.

We show that freeze-dried actin filaments which have been rotary shadowed with a light coat of platinum appear very similar in morphology and width to negatively-stained filaments. The addition of a thicker coat of platinum to such preparations gives the actin filaments a different morphology and width, which are similar to those of the rotary-shadowed, quick-frozen filaments described by Heuser and Kirschner (J. Cell Biol. 1980, 86:212-234). The consistent view of the actin filament presented here, particularly its 7-8-nm width, can be interpreted in terms of the overall orientation of the actin subunit in the actin filament.

Actins↗
PubMed1983

The fibrillar substructure of keratin filaments unraveled.

We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four "4.5-nm protofibrils" can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into "2-nm protofilaments." In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils.

Cations, Divalent↗
PubMed1982

Actin and myosin function in acanthamoeba.

We have studied the functions of contractile proteins in Acanthamoeba by a combination of structural, biochemical and physiological approaches. We used electron microscopy and image processing to determine the three-dimensional structure of actin and the orientation of the molecule in the actin filament. We measured the rate constants for actin filament elongation and re-evaluated the effect of MgCl2 on the filament nucleation process. In Acanthamoeba actin polymerization is regulated, at least in part, by profilin, which binds to actin monomers, and by capping protein, which both nucleates polymerization and blocks monomer addition at the 'barbed' end of the filament. To test for physiological functions of myosin-II, we produced a monoclonal antibody that inhibits the actin-activated ATPase. When microinjected into living cells, this active-site-specific antibody inhibits amoeboid locomotion. We expect that similar experiments can be used to test for the physiological functions of the other components of the Acanthamoeba contractile system.

Actins↗
PubMed1982

Brain spectrin, a membrane-associated protein related in structure and function to erythrocyte spectrin.

An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes. This protein co-sediments with and cross-links actin filaments, associates with spectrin-binding sites on erythrocyte membranes, and has been visualized by rotary shadowing as an extended, flexible rod. The brain spectrin comprises 3% of the total membrane protein, and may have a major role in mediating linkage of actin to membranes.

Actins↗
PubMed1982

Three-dimensional structure of proteins determined by electron microscopy.

Recent developments in specimen preparation and image processing techniques have made it possible to determine the three-dimensional structure of proteins by electron microscopy. Periodic supramolecular aggregates of the protein under investigation are requiring to minimize radiation damage and to maximize the signal-to-noise ratio of structural detail. Useful information about the fine structure of the protein (e.g. binding sites for interacting molecules, antigenic determinants) can often be obtained by stoichiometric labeling of the ordered arrays with interacting molecules or antibody fragments, and computing difference maps from the reconstructions of the labeled and native structures. The use of this approach to molecular structure determination of proteins will be discussed in light of our work with bacteriophage and actin.

Actins↗
PubMed1982

Polymorphism of actin paracrystals induced by polylysine.

We describe a method for the induction of different polymorphic forms of actin filament paracrystals. This polymorphism is probably based on differences in the stagger and/or polarity of adjacent filaments in single-layered paracrystals and by superposition of different layers in multilayered paracrystals. The helical parameters defining the filament geometry are indistinguishable for the different polymorphic forms observed and for the four different actins used. Analysis of these paracrystals, some of which are ordered to better than 2.5 nm, should provide a reference structure suitable for alignment and orientation within the actin filament of high resolution models of the actin monomer obtained from crystal data.

Actins↗