Aprotinin binding to amyloid fibrils.

Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, beta-amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot-blot ligand-binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of beta-sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand beta-sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.     

Intravital study of the hydrophobic interactions in protein fibrils by a polarization-fluorescence method. I. Collagen fibrils

It has been shown earlier that anisotropy of extrinsic fluorescence (AEF) of ordered structures in living cell may serve as a measure of the free structure energy. The experimental study of AEF changes of myelin at varying temperatures (0-30 degrees C) revealed cold denaturation. This phenomenon occurs only in cases when the structure in question is stabilized through hydrophobic interactions. The purpose of this work was to show that not only membranes but also some native ordered protein structures might be analysed by fluorescence polarization to detect hydrophobic interactions. The fluorescence anisotropy of primulin adsorbed by collagen fibrils from rat tail has been investigated. The fluorophore orientation decreases with temperature, i.e. here the phenomenon of cold denaturation is observed. When the medium humidity falls down to 80%, no cold denaturation occurs. AEF of collagen fibrils depends to a considerable extent on organic substances (ethanol, ethylene glycol) added in small concentrations to the incubation medium. It is concluded that the dependence of the whole collagen molecular structure in fibrils on the content and structure of the solvent shell is of critical nature.     

Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

Purification and preliminary characterization of Spiroplasma fibrils.

Fibrils 3.5 nm in diameter were released from the honeybee spiroplasma (BC3) by treatment with detergents and then purified by isopycnic centrifugation. Purified fibrils were flexuous, of indeterminate length, and had an axial repeat of 8.5 nm. The fibrils were associated in pairs, but in 1 M salt formed aggregates with a marked striated appearance. Pronase completely degraded the fibrils, but trypsin had little effect. The fibrils were composed of a single protein of molecular weight 55,000 which represented about 1% of the total cell protein. A protein of molecular weight 26,000 appeared to be associated with the fibrils. The significance of this in relation to membrane attachment and the possible role of fibrils in maintenance of cell shape and in motility are discussed.     

Stimulation of glycosaminoglycan biosynthesis by amyloid fibrils.

Since amyloid-laden organs have an increased glycosaminoglycan content, the effect of amyloid fibrils on glycosaminoglycan metabolism by normal fibroblasts was examined. In comparison with controls, synthesis of glycosaminoglycans, primarily hyaluronic acid, was increased by an average of 48 and 93% respectively when 0.1 and 1.0 mg of amyloid fibrils/ml was added to the cultures.     

Amyloid fibrils

Amyloid refers to the abnormal fibrous, extracellular, proteinaceous deposits found in organs and tissues. Amyloid is insoluble and is structurally dominated by β?sheet structure. Unlike other fibrous proteins it does not commonly have a structural, supportive or motility role but is associated with the pathology seen in a range of diseases known as the amyloidoses. These diseases include Alzheimer’s, the spongiform encephalopathies and type II diabetes, all of which are progressive disorders with associated high morbidity and mortality. Not surprisingly, research into the physicochemical properties of amyloid and its formation is currently intensely pursued. In this work we will highlight the key scientific findings and discuss how the stability of amyloid fibrils impacts on bionanotechnology.     

Spiroplasma fibrils

Spiroplasmas contain long flexuous fibrils composed of a protein, molecular weight 55,000, which is specific to Spiroplasma and is highly conserved among different species. The protein cannot be detected in other wall-less prokaryotes reported to contain actin-like proteins and is unrelated to eukaryotic cytoskeletal components. Fibrils occur in similar concentrations in helical and nonhelical strains of Spiroplasma citri. Proposals that fibrils are responsible for maintenance of helical cell shape and rotary motility are discussed in the light of these findings. Evidence is presented which suggests that fibrils may be arrayed as one or more bundles in intact cells and a consistent association of these structures with DNA filaments is noted. These observations are discussed in relation to possible models to account for the maintenance of helical morphology and to the segregation of chromosomes during cell division.     

Involvement of periplasmic fibrils in motility of spirochetes.

Nonmotile (Mot-) strains of Spirochaeta aurantia and Spirochaeta halophila were isolated with a procedure involving mutagenesis of motile wild-type cells. Electron microscopy showed that a Mot- mutant strain of S. halophia possessed incomplete periplasmic fibrils, inasmuch as most or all of the filamentous portion of the periplasmic fibrils was absent. Some of the cells of this Mot-, fibril-defective mutant strain lacked the filamentous portion of the periplasmic fibrils and formed proximal hooks, whereas other cells appeared to have a very small segment of the filamentous portion of the periplasmic fibrils attached to the proximal hooks. Motile revertants were isolated repeatedly from cultures of the Mot-, fibril-defective mutant and from S. halophila Mot- mutants that completely lacked periplasmic fibrils. The motile revertants possessed periplasmic fibrils ultrastructurally indistinguishable from wild-type periplasmic fibrils. This study indicates that periplasmic fibrils play an essential role in the motility of spirochetes.     

Structural domains of the tight junctional intramembrane fibrils.

Freeze-fracture reveals intramembrane fibrils lying along the intermembrane contacts that characterize tight junctions. Tight junctions from a variety of species are reexamined here by rapid freezing prior to freeze-fracture. The tight junction fibril is uprooted alternatively from either the cytoplasmic or the exoplasmic hemibilayer during freeze-cleavage, exposing two distinct but complementary views of its hybrid structure within the same replica. When the transmembrane fibril is uprooted from the exoplasmic hemibilayer it appears on the P-fracture face as a smooth-surfaced cylinder which is sometimes resolved into periodic globular structures. The lack of indication that the P-face cylinder has been pulled out through the opposite membrane half indicates that this domain of the fibril is, in large part, buried in the hydrophobic interior of the membrane. However, when the transmembrane fibril is uprooted from the cytosolic hemibilayer it appears on the E-fracture face as a row of irregular intramembrane particles. The irregular particles on the E-face aspect of the fibril are interpreted as corresponding to transmembrane protein segments that may very well make projections onto the cytosolic surface of the bilayer. En face views of the outermost junction strand between adjacent epithelial cells show periodic lines on the bilayer on each side of the junction which are interpreted as periodic transmembrane protein segments arising from the core structure of the tight junction fibril. If the backbone of the tight junction strand is an inverted cylindrical micelle, it must typically include proteins, which might anchor it to structures outside the membrane bilayer.     

The crystalline structure of collagen fibrils in tendon.

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with $\text{b = 75.5}\text{A}\text{?}$, β = 93 °, $\text{a =}\text{b}\text{sin}\text{β}$, a = 90 °, $\text{c = n × 668}\text{A}\text{?}$, where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.     

Plasticity of amyloid fibrils

In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Abeta (1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology. This view links amyloid with the prehistorical and 20th century use of proteins as starting materials to make films, fibers, and plastics, and with the classic protein fiber stretching experiments of the Astbury group. Viewing amyloids from the point of view of the polymer chemist may shed new light on a number of issues, such as the role of protofibrils in the mechanism of amyloid formation, the biological potency of fibrils, and the prospects for discovering inhibitors of amyloid fibril formation.