The fibril structure of type V collagen triple-helical domain

Although the triple-helical structure of fibrillar collagen is regarded in general as being quite similar, each type of collagen molecule has inherent characteristics in the triple-helical domain. Few studies have ever been performed in terms of the aggregate structure of the triple-helical domain of fibrillar collagen. Reconstituted aggregates from the purified triple-helical domain of each type of fibrillar collagen might amplify the subtle differences in the structural characteristics of each type of collagen molecule. In this study, the reconstituted aggregate structure of pepsin-treated type V collagen (type Vp collagen), that is, virtually its triple-helical domain was characterized by transmission electron microscopy. Pepsin-treated type I (type Ip) and type II (type IIp) collagen were compared with type Vp collagen. Unique features of the aggregate structure of the triple-helical domain of the type V collagen can be summarized as follows:These results suggested that the lateral packing of the triple-helical domain of type V collagen is determined by its molecular structure. The characteristics of type Vp collagen fibrils might be explained by their characteristic amino acid composition. A significant feature of the triple-helical domain of type V collagen is the high content of glycosylated hydroxylysine residues. Molecular model building of the collagenous structure suggests that a change in surface roughness is conspicuous by incorporating the glycosylated hydroxylysine residues. More than a ten-fold content of bulky glycosylated hydroxylysine residues in type V collagen compared to that of type I might have a significant influence on both the intermolecular and interfibrillar interactions of the triple-helical domain of type V collagen molecule.     

Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly

Type V collagen is a quantitatively minor fibrillar collagen with a broad tissue distribution. The most common type V collagen isoform is alpha1(V)(2) alpha2(V) found in cornea. However, other isoforms exist, including an [alpha1(V)alpha2(V)alpha3(V)] form, an alpha1(V)(3) homotrimer and hybrid type V/XI forms. The functional role and fibrillar organization of these isoforms is not understood. In the cornea, type V collagen has a key role in the regulation of initial fibril assembly. Type I and type V collagen co-assemble into heterotypic fibrils. The entire triple-helical domain of the type V collagen molecules is buried within the fibril and type I collagen molecules are present along the fibril surface. The retained NH(2)-terminal domains of the type V collagen are exposed at the surface, extending outward through the gap zones. The molecular model of the NH(2)-terminal domain indicates that the short alpha helical region is a flexible hinge-like region allowing the peptide to project away from the major axis of the molecule; the short triple-helical regions serve as an extension through the hole zone, placing the tyrosine-rich domain at the surface. The assembly of early, immature fibril intermediates (segments) is regulated by the NH(2)-terminal domain of type V collagen. These NH(2)-terminal domains alter accretion of collagen molecules onto fibrils and therefore lateral growth. A critical density would favor the initiation of new fibrils rather than the continued growth of existing fibrils. Other type V collagen isoforms are likely to have an important role in non-cornea tissues. This role may be mediated by supramolecular aggregates different from those in the corneal stroma or by an alteration of the interactions mediated by tissue-specific type V collagen domains generated by different isoforms or aggregate structures. Presumably, the aggregate structure or specific domains are involved in the regionalization of fibril-associated macromolecules necessary for the tissue-specific regulation of later fibril growth and matrix assembly stages.     

Influence of saline and pH on collagen type I fibrillogenesis in vitro: fibril polymorphism and colloidal gold labelling

We have produced different collagen type I fibrils by in vitro fibrillogenesis of acetic acid-soluble collagen within the pH range 2.5-9.0, in the presence and absence of 150 mM NaCl. The varying relatively stable molecular assemblies and polymorphic fibrillar end-products produced after 24 h incubation have been assessed and compared by the TEM study of specimens negatively stained with uranyl acetate. In the presence of 150 mM NaCl, the assembly of collagen at low pH (2.5) leads to the formation of initial molecular aggregates that progressively link together at slightly higher pH (5.0) to form sub-fibrils and spindle-shaped D-banded bundles of sub-fibrils. At pH 6.0 these D-banded bundles aggregate into larger spindle-shaped fibrils with lateral misalignment of the D-banding across the bundle. However, at pH 7.0 and 8.0, in the presence of 150 mM NaCl, the characteristic parallel-sided mature D-banded collagen type I fibres are formed. At pH 9.0 more loosely formed parallel-sided D-banded collagen fibrils are present, within which the spindle-shaped sub-fibrils can be defined by negative staining more convincingly than at pH 7-8. In the presence of 50 mM buffer at pH 2.5, but absence of 150 mM NaCl, collagen type I forms disorganized periodic initial molecular aggregates, which have a tendency to link together to form sub-fibrils. Flexuous collagen type I sub-fibrils predominate at pH 5.0, alongside large spindle-shaped fibrils that possess a regular transverse approximately 10 nm periodicity, with an oblique approximately 67 nm periodicity, significantly different to the D-banding periodicity. At pH 7.0 and pH 8 in the absence of saline loosely-formed flexuous and spindle-shaped fibres co-exist, with underlying sub-fibrils visible, but at pH 9.0 only disorganized flexuous fibrillar aggregates are present. Colloidal gold labelling of the characteristic D-banded collagen type I fibrils with 5 nm and 2 nm chemically reactive gold particles reveals a periodic labelling pattern, which is not apparent with 10 nm and 15 nm gold particles, due to steric hindrance. The flexuous and spindle-shaped collagen fibrils also bind 2 nm gold particles in a specific manner. In all cases, the specific chemisorption of gold onto the collagen fibrils is probably determined by the availability of repeating amino acid side chains of the collagen molecules along the fibril surface. The controlled production of varying stable collagen type I fibrillogenesis products is likely to be of value within numerous areas of biotechnology, biology and medicine, including experimental biomineralization.     

Structural variations of collagen in normal and pathological tissues: role of electron microscopy

The spectrum of ultrastructural appearances assumed by collagen in normal and pathological tissues is illustrated using techniques of thin section transmission electron microscopy and computer-assisted analysis. The normal fibrillar collagen types are described in order to provide a basis for comparing other normal and abnormal forms. In normal tissues, the anchoring fibril and basal lamina (basement membrane) represent tissue structures largely containing collagen but differing significantly in organisation from normal types I to III fibrillar collagen. In pathological tissue, deviations from normal fine structure are reflected in abnormal aggregates of collagen fibrils (amianthoid and skeinoid fibres) and abnormalities in fibril diameter and cross-sectional profile. Fibrous and segment long-spacing collagen represent two further organisational variants of collagen, the former found widely in pathological tissues, the latter very rarely. Much remains to be discovered about these abnormal collagen variants-their mode of formation, the cells that produce them, and their roles. They also present a challenge for the collagen biologist formulating hypotheses of collagen fibril assembly and molecular organisation.     

Fluorescence Investigation of Interactions Between Novel Benzanthrone Dyes and Lysozyme Amyloid Fibrils

A series of novel fluorescent benzanthrone dyes have been tested for their ability to identify and characterize fibrillar aggregates of lysozyme prepared by protein denaturation in concentrated ethanol solution (F_eth) or acidic buffer (F_ac). Quantitative parameters of the dye association with native and fibrillar protein have been derived from the results of fluorimetric titration. The binding characteristics proved to be different for F_eth- and F_ac-bound benzanthrones, highlighting the dye sensitivity to the distinctions in fibril morphology. By comparing the dye preference to fibrillar protein aggregates, AM2, A8 and A6 were selected as the most prospective amyloid tracers. Based on the analysis of red edge excitation shifts and fluorescence lifetimes of the amyloid-bound dyes it was assumed that surface grooves or dry “steric zipper” interface are potential fibril binding sites for the novel fluorophores.     

Fourier transform of the collagen triple-helical structure and its significance

The Fourier transforms of the collagen molecular structure have been calculated taking into consideration various side chain atoms, as well as the presence of bound water molecules. There is no significant change in the calculated intensity distribution on including the side chain atoms of non-imino-acid residues. Taking into account the presence of about two bound water molecules per tripeptide unit, the agreement with the observed x-ray pattern is slightly improved. Fourier transforms have also been calculated for the detailed molecular geometries proposed from other laboratories. It is found that there are no major differences between them, as compared to our structure, either in the positions of peak intensity or in the intensity distribution. Hence it is not possible to judge the relative merits of the various molecular geometries for the collagen triple helix from a comparison of the calculated transforms with the meagre data available from its x-ray fibre pattern. It is also concluded that the collagen molecular structure should be regarded as a somewhat flexible chain structure, capable of adapting itself to the requirements of the different side groups which occur in each local region.     

Antibacterial and Miscibility Properties of Chitosan/Collagen Blends

The miscibility, bioactivity, and antibacterial properties of chitosan/collagen specimens were systematically studied. The specimens were prepared by blending collagen and chitosan with varying deacetylation degrees in solutions; the collagen molecules had been extracted from pigskins using the acid swelling-pepsin digestion method. To understand the miscibility properties of collagen and chitosan molecules, the intrinsic viscosity and differential scanning calorimetry analysis of collagen, chitosan, and collagen/chitosan specimens were performed. The instrinsic viscosity measurements suggested that chitosan and collagen molecules with varying deacetylation degrees were miscible at molecular level for all compositions and degrees of deacetylation of chitosan/collagen mixture solutions prepared in this study. Fourier transform infrared analyses suggested that the percentage of preserved triple helix structures present in collagen molecules in collagen/chitosan specimens decreased with increasing chitosan contents, since the ratios of peak absorbance at 1239 cm^?1 of amide III and 1455 cm^?1 of C?H bending of collagen/chitosan specimens decreased significantly with increase in their chitosan contents. Abnormally high denaturation temperatures (T_d) were observed as the chitosan contents of collagen/chitosan specimens reached 40 wt%, at which T_d of collagen molecules was even higher than that of the corresponding pure chitosan molecules with varying deacetylation degrees. The antibacterial activity of collagen/chitosan blends increased consistently with increasing deacetylation degrees and concentrations of chitosan molecules in collagen/chitosan solutions. Possible explanations for these interesting thermal denaturation, antibacterial, and miscibility properties of chitosan/collagen specimens are reported.     

Self-aggregation of fibrillar collagens I and II involves lysine side chains

Several properties of fibrillar collagens depend on abundance and position of ionic amino acids. We recently demonstrated that N-methylation and N-acetylation of Lys/Hyl amino group did not significantly alter the thermal stability of the triple helical conformation and that the binding of modified collagens I and II to decorin is lost only on N-acetylation. The positive charge at physiological pH of Lys/Hyl side chains is preserved only by N-methylation. We report here the new aspect of the influence of the same modifications on collagen self-aggregation in neutral conditions. Three collagen preparations are very differently affected by N-methylation: acid-soluble type I collagen maintains the ability to form banded fibrils with 67-nm periodicity, whereas almost no structured aggregates were detected for pepsin-soluble type I collagen; pepsin-soluble type II collagen forms a very different supramolecular species, known as segment long spacing (SLS). N-acetylation blocks the formation of banded fibrils in neutral conditions (as did all other chemical modifications reported in the literature), demonstrating that the positive charge of Lys/Hyl amino groups is essential for self-aggregation. Kinetic measurements by turbidimetry showed a sizeable increase of absorbance only for the two N-methylated samples forming specific supramolecular aggregates; however, the derivatization affects aggregation kinetics by increasing lag time and decreasing maximum slope of absorbance variation, and lowers aggregation competency. We discuss that the effects of N-methylation on self-aggregation are caused by fewer or weaker salt bridges and by decrease of hydrogen bonding potential and conclude that protonated Lys side chains are involved in the fibril formation process.     

Characterisation of structural changes in collagen with Raman spectroscopy

Raman spectroscopy can detect conformational changes in collagen structures and these findings are reviewed in this article. More specifically, some progressive diseases are mainly caused by alterations of collagen molecules but what is occurring at the biochemical level of this complex molecule usually remains unclear. While it may be true that a number of analytical techniques can analyze collagen, most of them have a series of limitations that limit their applicability to a wide range of samples. To understand in more detail the progression of a disease due to changes in the collagen structure, a technique that can detect subtle alterations at the biochemical level is needed. Raman spectroscopy is a label-free and noninvasive technique that can easily pick up on any conformational changes reflected primarily at the lipids, amides and proline and hydroxyproline regions. This review is the first compilation of studies of conformational changes in collagen molecules, providing help to understand changes in collagen biochemistry that can be of relevance to the human wound healing, ageing and pathologies.     

Dynamics of hydrogen-bonded water networks under high pressure: Neutron scattering and computer simulation

Abstract

Molecular dynamics simulation of proton-ordered high pressure ice modifications II and IX was performed. Dynamics of both isotope varieties, H₂O and D₂O, was simulated. Rectangular simulation box of ice II contained 576 and that of ice IX 768 molecules. The average kinetic energy corresponded to 82 and 201 K for ice II and to 87 and 203 K for ice IX. One-phonon densities of states were calculated via Fourier transformation of velocity autocorrelation functions and compared with those found experimentally from inelastic incoherent neutron scattering. This characteristic was calculated for all the molecules, as well as for the molecules of a particular crystallographic type. Both simulated ice modifications contain molecules of two different structural types. Dynamic characteristics of molecules of different types are slightly different. Splitting of the librational peak at about 60–70meV observed in the ice II experimental spectrum is mainly due to such difference. In the case of ice II simulated spectra reproduce experimental ones quite reasonably in the whole range of energies, while in the case of ice IX agreement with the experiment is worse.     

Collagen fibril organisation in mammalian vitreous by freeze etch/rotary shadowing electron microscopy

Mammalian vitreous gel contains two major network-forming polymeric systems: long, thin fibrils comprising predominantly type II collagen and a meshwork of hyaluronan. The gel structure is maintained primarily by the collagen component, but little is known about the mechanisms of spacing of the collagen fibrils and of interactions between fibrils to form a stable network. In this study we have applied the technique of freeze etching/rotary shadowing electron microscopy in order to reveal the fibrillar network in central, cortical and basal vitreous and to understand the structural relationship between the collagen fibrils. The fibrils were arranged side by side in narrow bundles that frequently branched to link one bundle to another. Only a minor part of the fibrillar network consisted of segments that had a diameter of a single fibril (16.4nm mean diameter). In addition, three morphologically distinct filamentous structures were observed that appeared to form links within the collagen fibrillar network: short, single interlinking filaments of 7.0nm mean diameter, network-forming filaments of 6.7nm mean diameter, and longer filaments of 8.2nm mean diameter. All three types of filamentous structure were removed by digestion of the vitreous gels with Streptomyces hyaluronan lyase prior to freeze etching, indicating that these structures contain or are stabilised by hyaluronan. These filamentous structures may contribute to the structural stability of the vitreous gel.     

Tri- and Pentamethine Cyanine Dyes for Fluorescent Detection of α-Synuclein Oligomeric Aggregates

The pathogenesis of Parkinson’s disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10^?6 M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2–2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more “crescent-shape” molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.     

利用二维同步荧光相关光谱研究温度对胶原溶液中分子聚集行为的影响

采用恒波长同步荧光法和二维相关分析技术研究了不同浓度Ⅰ型胶原溶液中胶原分子聚集行为随温度升高(10~70 ℃)的变化规律。选取0.2,0.4,1.6 mg·mL-1的胶原溶液,在初始温度下各浓度溶液中胶原分子分别处于单分子状态、较低程度和较高程度的聚集态。研究表明：波长差为9 nm的同步荧光光谱中,激发波长282和292 nm处荧光峰分别归属于未参与形成氢键的Tyr(酪氨酸)残基和参与形成氢键的Tyr残基。对升温过程同步荧光数据进行二维相关分析,得两荧光值对温度的响应顺序,进而推测得到：当温度低于30 ℃时,0.2 mg·mL-1溶液中出现了胶原分子间形成Tyr残基参与的氢键的趋势。0.4和1.6 mg·mL-1的溶液中原有聚集体可能发生进一步聚集,形成疏水微区。当逐步接近胶原变性温度(36~38 ℃)时,推测0.4和1.6 mg·mL-1胶原溶液中的疏水微区和聚集体有被破坏的趋势,而0.2 mg·mL-1胶原溶液保持分子间形成氢键的趋势。超过胶原变性温度时,各浓度溶液中胶原分子三股螺旋结构发生松散。当超过45 ℃时,胶原分子三股螺旋结构松散的趋势更为明显。     

Polymorphism of amyloid-β fibrils and its effects on human erythrocyte catalase binding

The Alzheimer's amyloid-β (Aβ) peptide exists as a number of naturally occurring forms due to differential proteolytic processing of its precursor molecule. Many of the Aβ peptides of different lengths form fibrils in vitro, which often show polymorphisms in the fibril structure. This study presents a TEM based analysis of fibril formation by eighteen different Aβ peptides ranging in length from 5 to 43 amino acids. Spectrophotometric analysis of Congo red binding to the fibrillar material has been assessed and the binding of human erythrocyte catalase (HEC) to Aβ fibrils has also been investigated by TEM. The results show that a diverse range of Aβ peptides form fibrils and also bind Congo red. The ability of both Aβ 1–28 and Aβ 29–40 to form fibrils indicates that there are at least two fibril-forming domains within the full-length Aβ 1–40 sequence, the ability of many Aβ peptides to form Congo red-binding aggregates suggests that there may be up to 4 possible aggregation promoting domains. The binding of HEC was limited to Aβ forms containing residues 29–32. The differing capacities of fibrillar and ribbon-like structures may reflect the accessibility of the 29–32 region and suggest that HEC may be able to discriminate between different forms of Aβ fibrils.     

Reversible and irreversible denaturation processes in globular proteins: from collective to molecular spectroscopic analysis

Different spectroscopic techniques were applied for studying the structural properties of lysozyme in salt‐free aqueous solutions. The results of vibrational and Brillouin scattering measurements were compared to obtain both single‐molecule and collective properties of the solutions. The characterization of the protein system, from the conformation of the polypeptide chain to the exposure of side chains to the solvent and the arrangement of the solution network, was then achieved in the range 25–85 °C. Through the analysis of the indole breathing mode, a different environment for the six tryptophan residues of an unfolded lysozyme could be evidenced. Short and long exposures to high temperatures were used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. These different thermal treatments were applied to distinguish between the effects of global unfolding of the single molecule from those of self‐aggregation and gel formation. It has been observed that clusterization occurs at melting temperatures with slow kinetics; also, aggregates evolve from the completely unfolded state of the protein and lead to a sensitive increase in viscosity. This effect probably hinders any further conformational rearrangement of the molecules in the aggregate; thus as a consequence, the disordered structure of clusters does not change to give the β‐sheet organization, characteristic of filaments or fibrils. Copyright © 2011 John Wiley & Sons, Ltd.     

Ordering of hydrogen bonds in high-pressure low-temperature H2O

The near K-edge structure of oxygen in liquid water and ices III, II, and IX at 0.25 GPa and several low temperatures down to 4 K has been studied using inelastic x-ray scattering at 9884.7 eV with a total energy resolution of 305 and 175 meV. A marked decrease of the preedge intensity from the liquid phase and ice III to ices II and IX is attributed to ordering of the hydrogen bonds in the proton-ordered lattice of the latter phases. Density functional theory calculations including the influence of the Madelung potential of the ice IX crystal correctly account for the remaining preedge feature. Furthermore, we obtain spectroscopic evidence suggesting a possible new phase of ice at temperatures between 4 and 50 K.     

Theoretical conformation of proline oligomers

Minimum energy (vacuum) conformations of proline oligomers and poly-L-proline have been calculated. The left-hand helix of trans-polyproline II becomes stable at the tetramer, whereas the right-hand helix of cis-polyproline I is not established until at least the pentamer. The potential minima include values of ψ (ψ = 163° trans, ψ = 56° cis) which yield forms of the polymer that are virtually identical with polyproline I and II in the solid state. Calculated repeat distances and residues per turn for the two forms are 1.90 Å and 3.18 (pp I) and 3.11 Å and 3.00 (pp II), compared with experimental values of 1.90 Å and 3.33 (pp I) and 3.12 Å and 3.00 (pp II). A third form, not previously described, is found to be almost equally energetically favorable with the trans form of polyproline II. This form, herein called polyproline III, is a trans left-hand helix with alternating rotation angles of 325° and 170°, and 3.35 residues per turn, which forms a right-hand superhelix. Using collagen II proline coordinates, this superhelix is found to have a translation of 8.87 Å with 8.5 residues per turn.     

Adsorptive parameters and influence of hot geometries on the SER(R) S spectra of methylene blue molecules adsorbed on gold nanocolloidal particles

The vibrational signatures of methylene blue (MB) dye molecule have been reported. Complete vibrational assignments of the molecule are presented for the first time. Concentration‐dependent surface enhanced resonance Raman scattering [SER(R) S] spectra of the molecule have been investigated. Fluorescence spectroscopic technique has been applied to explore the concentration of the probe molecule actually adsorbed on the gold nanocolloidal (AuNC) surface. The free energy of adsorption and saturated concentration of MB molecules on AuNC surface are also estimated. Gigantic enhancements ~10⁵–10¹³ orders of magnitude have been recorded for the enhanced Raman bands in the SER(R) S spectra. The possible orientation of the molecule on the AuNC surface and their probable protrusions within the hot spots have been suggested. The hot geometries and correlation between the plasmonic behavior of the nanoparticles and enhancement efficiencies of the SER(R) S band have been mapped with the aid of three‐dimensional finite difference time domain (3D‐FDTD) simulations. Application of soft lithographic technique to engineer the pattern formation of hetero dimeric spherical aggregates will be an interesting field of study in future to enhance the detection limit of this and similar types of dye molecules. Copyright © 2015 John Wiley & Sons, Ltd.     

Exploration of twisted intramolecular charge transfer fluorescence properties of trans-2-[4-(dimethylamino)styryl]benzothiazole to characterize the protein–surfactant aggregates

The characterization of aggregates of an anionic surfactant, sodium dodecyl sulphate (SDS) with bovine serum albumin (BSA) in various regions of binding isotherm of SDS to BSA with increasing concentration of the former have been done by exploring the twisted intramolecular charge transfer (TICT) fluorescence properties of a probe, trans-2-[4-(dimethylamino)styryl] benzothiazole (DMASBT). The TICT fluorescence, steady-state fluorescence anisotropy and time-resolved fluorescence of DMASBT, and the fluorescence resonance energy transfer (FRET) study reveal the characteristics of the native protein as well as the protein–surfactant aggregates viz., micropolarity, microviscosity, locations of probe, denaturation of protein in various regions of binding isotherm, and also the validation of necklace-bead model. The changes in the polarity and the viscosity of the microenvironment around the probe from one binding region of SDS to other have been reflected in the highly sensitive fluorescence properties of DMASBT. The study of FRET between the DMASBT and the tryptophan residue (Trp) of BSA has identified the locations of the probe molecule in the native protein as well as that in various BSA–SDS aggregates. The energy transfer efficiency decreases, whereas the distance between the DMASBT and the Trp residue increases with increasing concentration of SDS. The significant change in the conformations of protein molecules during the non-cooperative binding region of SDS is evidenced by the fluorescence anisotropic behavior of DMASBT in the same region.