Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy (original) (raw)
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Fibrous Long Spacing Collagen Ultrastructure Elucidated by Atomic Force Microscopy
Biophysical Journal, 1998
Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of ␣ 1 -acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically ϳ150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.
Protein Science, 2019
Collagen fibrils represent a unique case of protein folding and self-association. We have recently successfully developed triple-helical peptides that can further selfassemble into collagen-mimetic mini-fibrils. The 35 nm axially repeating structure of the mini-fibrils, which is designated the d-period, is highly reminiscent of the wellknown 67 nm D-period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo-identical repeating sequence units in the primary structure of the designed peptides that give rise to the d-period of the quaternary structure of the mini-fibrils. In this work, we characterize the selfassembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple-helix domain of Col877 consists of three pseudo-identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 selfassociate laterally under fibril forming conditions to form mini-fibrils having the predicted d-period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self-assembly of collagen mini-fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen-mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications. K E Y W O R D S axial-repeating structure of protein, collagen-mimetic fibrils, fibrous molecular assembly, protein design: fibrous protein, self-association of collagen triple helices, self-association of protein, sequence periodicity and protein structure, tailored functional collagen-mimetic biomaterial
Self-aggregation of fibrillar collagens I and II involves lysine side chains
Micron, 2006
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 Nmethylated 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 selfaggregation 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. #
Structural investigations on native collagen type I fibrils using AFM
Biochemical and Biophysical Research Communications, 2007
This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.
STEM/TEM studies of collagen fibril assembly
Micron, 2001
Quantitative scanning transmission electron microscopy (STEM), implemented on a conventional transmission electron microscope with STEM-attachment, has been a primary tool in our laboratory for the quantitative analysis of collagen fibril assembly in vivo and in vitro. Using this technique, a precise measurement of mass per unit length can be made at regular intervals along a fibril to generate an axial mass distribution (AMD). This in turn allows the number of collagen molecules to be calculated for every transverse section of the fibril along its entire length. All fibrils show a near-linear AMD in their tip regions. Only fibrils formed in tissue environments, however, show a characteristic abrupt change in mass slope along their tips. It appears that this tip growth characteristic is common to fibrils from evolutionarily diverse systems including vertebrate tendon and the mutable tissues of the echinoderms. Computer models of collagen fibril assembly have now been developed based on interpretation of the STEM data. Two alternative models have so far been generated for fibril growth by accretion; one is based on diffusion limited aggregation (DLA) and the other based on an interface-limited growth mechanism. Inter-fibrillar fusion can also contribute to the growth of fibrils in vertebrate tissues and STEM data indicates the presence of a tight regulation in this process. These models are fundamental for the hypotheses regarding how cells synthesise and spatially organise an extracellular matrix (ECM), rich in collagen fibrils. ᭧
Evidence of a Discrete Axial Structure in Unimodal Collagen Fibrils
Biomacromolecules, 2011
The collagen fibrils of cornea, blood vessel walls, skin, gut, interstitial tissues, the sheath of tendons and nerves, and other connective tissues are known to be made of helically wound subfibrils winding at a constant angle to the fibril axis. A critical aspect of this model is that it requires the axial microfibrils to warp in an implausible way. This architecture lends itself quite naturally to an epitaxial layout where collagen microfibrils envelop a central core of a different nature. Here we demonstrate an axial domain in collagen fibrils from rabbit nerve sheath and tendon sheath by means of transmission electron microscopy after a histochemical reaction designed to evidence all polysaccharides and by tapping-mode atomic force microscopy. This axial domain was consistently found in fibrils with helical microfibrils but was not observed in tendon, whose microfibrils run longitudinal and parallel.
Nanoscale measurements of the assembly of collagen to fibrils
International Journal of Biological Macromolecules, 2010
Observing the self-assembly of collagen from single collagen monomers to higher order fibrils and fibers provides a bottom-up approach to engineering its ultrastructure in comparison to structural studies of already formed collagen fibers. This approach can be used for the fabrication of controlled collagen-based biomaterials with varying mechanical properties. Here, we investigate the time-dependent self-assembly of collagen into single fibrils in vitro through high resolution imaging of collagen type 1 prior to fibrillogenesis. This was confirmed by comparing persistence length and diameter in controlled experiments and studying the morphology and mechanical properties of nanoscale collagen fibrils through AFM nanoindentation measurements. The Young's modulus of these collagen fibrils was estimated to be around 1 GPa in the dehydrated state. The stability and mechanical characteristics of collagen obtained in these experiments indicate the hierarchical assembly occurs at both a structural and mechanical level.
Different architectures of the collagen fibril: morphological aspects and functional implications
International Journal of Biological Macromolecules, 1989
Several tissues known to contain collagen fibrils with a 'helical' arrangement were studied by t.e.m. and freeze-fracture. In all the tissues examined, the diameter of the collagen fibrils appeared to be tissue-specific and fairly constant within the same tissue. No statistical differences, on the contrary, were detectable in the coiling angle which appeared similar in all the tissues and independent of both diameter and age of the fibril. Rat tail tendon was also examined under the same technical conditions and showed collagen fibrils of large and very heterogeneous diameter and with a consistent 'straight' arrangement. These data seem to suggest that the 'helical' and 'straight' arrangements may actually identify different types of collagen fibrils. The authors discuss the possible functional significance of these arrangements and present two hypotheses on the three-dimensional structure of the 'helical' fibril.
Collagen Fibrillogenesis in Tissues, in Solution and from Modeling: A Synthesis
Journal of Investigative Dermatology, 1982
Collagen fibril formation has been studied in tissues by light and electron microscopy; in solution by light scattering and microscopy; and from modeling based on the amino acid sequence of type I collagen. Taken to gether these studies indicate that collagen fibril assem bly involves a stepwise formation of intermediate aggre gates in which each intermediate is formed from earlier aggregates. In this sequence, monomeric collagen con tributes only to the formation of early aggregates; and fibrils grow in length by the addition of intermediate aggregates to the end of a subfibril and in width by lateral wrapping of subfibrils. Modeling based on amino acid sequence data of possible intermolecular charge charge interactions indicate 2 different kinds, one which promotes linear aggregation and the other which pro motes lateral aggregation. The effects of different colla gens and coprecipitants such as glycoproteins and pro teoglycans can begin to be explained by their influence on the character of intermediate subassemblies. Ultra structural data from 2 tissues, embryonic cornea and tendon, indicate that the site of fibril growth and assem bly is at the cell surface.