Methods for structural characterization of prefibrillar intermediates and amyloid fibrils

Protein fibrillation is first and foremost a structural phenomenon. Adequate structural investigation of the central conformational individuals of the fibrillation process is however exceedingly difficult. This is due to the nature of the process, which may be described as a dynamically evolving equilibrium between a large number of structural species. These are furthermore of highly diverging sizes and present in very uneven amounts and timeframes. Different structural methods have different strengths and limitations. These, and in particular recent advances within solution analysis of the undisturbed equilibrium using small angle X-ray scattering, are reviewed here.     

Conformational study and hydrogen bonds detection on elastin-related polypeptides using X-ray photoelectron spectroscopy

The chemical bonds of the pentapeptide sequence of elastin ValGlyGlyValGly (VGGVG), both in its monomer and polymer forms, were correlated with their XPS spectra through a well-established curve-fitting procedure. To aid in this correlation, the C1s, O1s, and N1s chemical shifts of the Boc-VGGVG-OEt, were validated by theoretical calculations, performed in the framework of the Koopman approximation of HF/6-31G molecular orbitals, leading to the "preferred" conformation of the protected monomer. Then the same curve-fitting procedure was adopted for interpreting the XPS spectra of the polypentapeptide as a powder, and the XPS results obtained both for monomer and polymer compounds were compared with those obtained by FT-IR. The polymer was then analyzed after deposition onto a silicon substrate, Si(100), either from methanol or water suspensions and the presence of hydrogen bonds was detected at the polymer/substrate interface and between the polymer chains. The "surface rearrangement" that could be inferred from XPS results strongly confirms that derived from AFM images previously obtained under the same experimental conditions. In particular, the observed amyloid conformation is stabilized by hydrogen bonds to water molecules included in the structure while the formation of the beaded string structure observed in deposits from methanolic suspension is probably mediated by hydrogen bonds to the hydrated silicon surface.     

Tau protein assembles into isoform- and disulfide-dependent polymorphic fibrils with distinct structural properties

Tauopathies are neurodegenerative diseases in which insoluble fibrillar aggregates of a microtubule-binding protein, Tau, are abnormally accumulated. Pathological Tau fibrils often exhibit structural polymorphisms that differ among phenotypically distinct tauopathies; however, a molecular mechanism to generate polymorphic Tau fibrils remains obscure. Here, we note the formation of a disulfide bond in isoforms of full-length Tau and show that the thiol-disulfide status as well as the isoform composition determines structural and morphological properties of Tau fibrils in vitro. Mainly two regions in a Tau primary sequence are found to act as structural blocks for building a protease-resistant core of Tau fibrils. Interactions among those two blocks for building a core structure depend upon the thiol-disulfide status in each isoform of Tau, which results in the formation of polymorphic fibrils with distinct structural properties. Furthermore, we have found that more diverse structures of Tau fibrils emerge through a cross-seeded fibrillation between heterologous pairs of Tau isoforms. We thus propose that isoform- and disulfide-dependent combinatorial interactions among multiple regions in a Tau sequence endow Tau fibrils with various structures, i.e. polymorphism.     

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.     

Collagen fibrils and elastic fibers in rat-tail tendon: an electron microscopic investigation

Earlier studies by the authors showed that the collagen fibrils in rat-tail tendon have a bi-modal distribution of fibril diameters from a time shortly after birth through to the onset of maturity at about 3–4 months. Present work has extended those observations for rats up to the age of 2 years. Histograms of the fibril diameter distributions for mature tail tendon and direct electron microscope observations show that the fibrils break down as the tendon ages. Further work on the constant diameter subfibrils of diameter 140 Å described previously, has confirmed that these are part of the elastic fibers present in tendon at all ages. It has been shown that there is relatively little variation in the collagen fibril diameter distribution as a function of the position of the specimen in the tail, and as the measured percentage of the area taken by the collagen fibrils present at any particular point. Estimation of the fibrillar collagen content of rat-tail tendon as a function of age indicates that it increases steadily from birth and reaches a maximum at the onset of maturity, beyond which the fibrillar collagen content appears to remain constant.     

Physical and structural basis for polymorphism in amyloid fibrils

As our understanding of the molecular structures of amyloid fibrils has matured over the past 15 years, it has become clear that, while amyloid fibrils do have well‐defined molecular structures, their molecular structures are not uniquely determined by the amino acid sequences of their constituent peptides and proteins. Self‐propagating molecular‐level polymorphism is a common phenomenon. This article reviews current information about amyloid fibril structures, variations in molecular structures that underlie amyloid polymorphism, and physical considerations that explain the development and persistence of amyloid polymorphism. Much of this information has been obtained through solid state nuclear magnetic resonance measurements. The biological significance of amyloid polymorphism is also discussed briefly. Although this article focuses primarily on studies of fibrils formed by amyloid‐β peptides, the same principles apply to many amyloid‐forming peptides and proteins.     

The electrophysiological properties of the structural polypeptides of the foot-and-mouth-disease virus]

The methodological approaches of isolation of preparations of FMDV structural polypeptides to analyse them by the electrophoresis and electro-focussing methods are presented. The value of isoelectric points of protein coat of FMDV structural polypeptides and corresponding them values of electric potential are determined. The similarity and differences of FMDV serotypes, characterized by the value of relative surface, falling on separate polypeptides, are determined for the virion structure on the basis of superposition principle. FMDV has been shown to possess the summarized negative charge of different values. The charge depends on the virus type and it is a determining condition for viruses resistance in environment. A graphical model of FMDV is suggested on the basis of systemic approach and it reflects the dipole character of electric charge distribution in virion structure and agrees with the virus model, built on the basis of icosahedron symmetry.     

Synthesis and structural characterization of silk-like materials incorporated with an elastic motif

Genetic engineering strategies were applied to synthesize silk-like materials, [(GVPGV)(2)GG(GAGAGS)(3)AS](n). The primary structure of these materials represents the repetitive crystalline region of Bombyx mori silk fibroins incorporated with an elastic motif selected from animal elastin. The oligonucleotides were designed to encode the desired recombinant proteins and then expressed in the Escherichi coli system. The expression and purification conditions for the production of the recombinant proteins were optimized. (13)C CP/MAS NMR was used for structural characterization in the solid state, where the isotope labeling was performed using a modified M9 medium. The secondary structures of these materials are primarily governed by the designated amino acid sequence, where the B. mori silk fibroin block, (GAGAGS)(3), tends to form the crystalline region, which is interrupted by the flexible (GVPGV)(2) block. The CD data suggested that the structure of these materials was length-dependent in the solution state, i.e., a higher molecule weight leads to a higher ordered structure.     

Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus.

Purified virions or radiolabeled lysates of infected MA104 cells were used to characterize the structural and nonstructural polypeptides of a porcine group C rotavirus. At least six structural proteins were identified from purified group C rotavirus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these, two (37,000- and 33,000-molecular-weight polypeptides) were associated with the outer shell, as demonstrated by the ability of EDTA to remove them from the purified virion. The other four polypeptides (molecular weights, 125,000, 93,000, 74,000, and 41,000) were located in the inner shell. The structural or nonstructural nature of a 25,000-molecular-weight protein identified in our studies was unclear. Glycosylation inhibition studies with tunicamycin in infected cells demonstrated that the 37,000- and 25,000-molecular-weight proteins were glycosylated and contained mannose-rich oligosaccharides identified by radiolabeling of the infected cells with [3H]mannose. The 37,000-molecular-weight outer shell glycoprotein was shown by pulse-chase experiments to be posttranslationally processed. The kinetics of viral polypeptide synthesis in infected cells were also studied, and maximal synthesis occurred at 6 to 9 h postinfection. The 41,000-molecular-weight inner capsid polypeptide was the most abundant and was the subunit structure of a 165,000-molecular-weight protein aggregate. Two polypeptides (molecular weights, 39,000 and 35,000) appeared to be nonstructural, as determined by comparison of the protein pattern of radiolabeled infected cell lysates with that of purified virions. Radioimmunoprecipitation was used to examine the serologic cross-reactions between the viral polypeptides of a group C rotavirus with those of a group A rotavirus. No serologic cross-reactivities were detected. The polypeptides of group A and C rotaviruses are compared and discussed.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Identification and characterization of sORF-encoded polypeptides

Abstract

Molecular biology, genomics and proteomics methods have been utilized to reveal a non-annotated class of endogenous polypeptides (small proteins and peptides) encoded by short open reading frames (sORFs), or small open reading frames (smORFs). We refer to these polypeptides as s(m)ORF-encoded polypeptides or SEPs. The early SEPs were identified via genetic screens, and many of the RNAs that contain s(m)ORFs were originally considered to be non-coding; however, elegant work in bacteria and flies demonstrated that these s(m)ORFs code for functional polypeptides as small as 11-amino acids in length. The discovery of these initial SEPs led to search for these molecules using methods such as ribosome profiling and proteomics, which have revealed the existence of many SEPs, including novel human SEPs. Unlike screens, omics methods do not necessarily link a SEP to a cellular or biological function, but functional genomic and proteomic strategies have demonstrated that at least some of these newly discovered SEPs have biochemical and cellular functions. Here, we provide an overview of these results and discuss the future directions in this emerging field.     

Amyloid-like properties of Saccharomyces cerevisiae cell wall glucantransferase Bgl2p: Prediction and experimental evidences

Glucantransferase Bgl2p is a major conserved cell wall constituent described for a wide range of yeast species. In the baker''s yeast Saccharomyces cerevisiae it is the only non-covalently bound cell wall protein that cannot be released from cell walls by sequential SDS and trypsin treatment. It contains seven amyloidogenic determinants. Circular dichroism analysis and fluorescence spectroscopy with thioflavin T indicate the presence of β-sheet structures in Bgl2p isolates. Bgl2p forms fibrils, a process that is enforced in the presence of other cell wall components. Thus the data obtained is the first evidence for amyloid-like properties of yeast cell wall protein—glucantransferase Bgl2p.Key words: glucantransferase Bgl2p, cell wall amyloid-like protein     

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.     

Ab initio predictions of structural and elastic properties of struvite: contribution to urinary stone research

In the present work, we carried out density functional calculations of struvite--the main component of the so-called infectious urinary stones--to study its structural and elastic properties. Using a local density approximation and a generalised gradient approximation, we calculated the equilibrium structural parameters and elastic constants C(ijkl). At present, there is no experimental data for these elastic constants C (ijkl) for comparison. Besides the elastic constants, we also present the calculated macroscopic mechanical parameters, namely the bulk modulus (K), the shear modulus (G) and Young's modulus (E). The values of these moduli are found to be in good agreement with available experimental data. Our results imply that the mechanical stability of struvite is limited by the shear modulus, G. The study also explores the energy-band structure to understand the obtained values of the elastic constants.     

Ab initio predictions of structural and elastic properties of struvite: contribution to urinary stone research

In the present work, we carried out density functional calculations of struvite – the main component of the so-called infectious urinary stones – to study its structural and elastic properties. Using a local density approximation and a generalised gradient approximation, we calculated the equilibrium structural parameters and elastic constants C _ijkl . At present, there is no experimental data for these elastic constants C _ijkl for comparison. Besides the elastic constants, we also present the calculated macroscopic mechanical parameters, namely the bulk modulus (K), the shear modulus (G) and Young's modulus (E). The values of these moduli are found to be in good agreement with available experimental data. Our results imply that the mechanical stability of struvite is limited by the shear modulus, G. The study also explores the energy-band structure to understand the obtained values of the elastic constants.     

Isolation and characterization of cytoplasmic fibrils from treponemes

Electron microscopy of Triton X-100-treated whole cells of an oral treponeme, Treponema sp. strain E-21, revealed that six cytoplasmic fibrils (CFs) helically wound as a bundle in the cytoplasm. The CFs were isolated and purified by disruption and solubilization of the cells followed by CsCl density gradient centrifugation. The purified CF preparation contained mostly fibrils of about 9 nm in width and very small amounts of thinner strands of about 3 nm in diameter. The CFs were apparently seen to be a tubular structure, but the isolated CFs had narrowed sites of about 4-5 nm in width lacking lumen-like images, possibly representing twisted sites. Thus, the CF did not seem to be a tubular structure. The purified CFs were composed of one major 82 kDa protein and a few minor proteins. The CFs were destructed by treatment with proteases, 8 M urea or 4 M guanidine hydrochloride. Very low tyrosine content (0.76 mol %) and lack of methionine were characteristic features for the 82 kDa protein. The CF preparations from the other five treponemes including Treponema phagedenis and T. denticola also had 82 kDa proteins as a major component, and the 82 kDa proteins of all of the treponemes had a common antigen when examined by using antiserum against the 82 kDa protein from Treponema sp. strain E-21. Furthermore, the 82 kDa protein was demonstrated to be a principal component of the CFs of all the treponemes by immunoelectron microscopy.     

Carbamylation promotes amyloidogenesis and induces structural changes in Tau-core hexapeptide fibrils

Background

Carbamylation is a non-enzymatic post-translational modification (PTM), which involves the covalent modification of N-terminus of protein or ε-amino group of Lys. The role of carbamylation in several age-related disorders is well documented, however, the relationship between carbamylation and neurodegenerative disorders including Alzheimer's disease remains uncharted.

Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes.

Diacylglycerol, a biological membrane second messenger, is a strong perturber of phospholipid planar bilayers. It converts multibilayers to the reverse hexagonal phase (HII), composed of highly curved monolayers. We have used x-ray diffraction and osmotic stress of the HII phase to measure structural dimensions, spontaneous curvature, and bending moduli of dioleoylphosphatidylethanolamine (DOPE) monolayers doped with increasing amounts of dioleoylglycerol (DOG). The diameter of the HII phase cylinders equilibrated in excess water decreases significantly with increasing DOG content. Remarkably, however, all structural dimensions at any specific water/lipid ratio that is less than full hydration are insensitive to DOG. By plotting structural parameters of the HII phase with changing water content in a newly defined coordinate system, we show that the elastic deformation of the lipid monolayers can be described as bending around a pivotal plane of constant area. This dividing surface includes 30% of the lipid volume independent of the DOG content (polar heads and a small fraction of hydrocarbon chains). As the mole fraction of DOG increases to 0.3, the radius of spontaneous curvature defined for the pivotal surface decreases from 29 A to 19 A, and the bending modulus increases from approximately 11 to 14 (+/-0.5) kT. We derive the conversion factors and estimate the spontaneous curvatures and bending moduli for the neutral surface which, unlike the pivotal plane parameters, are intrinsic properties that apply to other deformations and geometries. The spontaneous curvature of the neutral surface differs from that of the pivotal plane by less than 10%, but the difference in the bending moduli is up to 40%. Our estimate shows that the neutral surface bending modulus is approximately 9kT and practically does not depend on the DOG content.