Decomposition of collagen type II fibrils in lamprey notochord viewed with TEM (A–D) and bovine articular cartilage (E and F).

<p>A) Native (prior to fixing) type II collagen fibrils, incubated in TBS as control for fibril samples shown in B–D. Average fibril size is around 35 nm. B) Collagen type II fibrils following short incubation with anti-biglycan antibody. Fibril diameter is 10–15 nm. C) Collagen type II fibrils following incubation in GHCl. Although severely disrupted, the fibril decomposition appears less complete than that of the antibody incubation (B). D) Collagen type II sample following mechanical disruption. Disruption of native fibril structure is highly localized, with large sections still intact. E) Native bovine articular cartilage (prior to fixing and staining for TEM). F) Bovine articular cartilage post 1 hour treatment with anti-biglycan. Black arrows point to thin-fibrils, white arrows point to normal sized thick-fibrils.</p

Model of antibody action on type II collagen fibrils.

<p>Coordinate models of the biglycan-type II collagen fibril complex based on the decoron-type I collagen fibril structures published recently (1) are shown with a model Fab (green) unit attaching to the biglycan (blue) epitope (colored red, A and ‘top’ view B). Because the epitope is located within a solvent filled channel of the collagen fibril <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032241#pone.0032241-Perumal1" target="_blank">[33]</a>, there is room for loops of the fab to dock with it, but its close proximately to the fibril-PG hydrogen bonding network located between the collagen fibril surface and the concave side of the PG-core proteins structure (1) may disrupt the positive interactions and dislodge the core protein from the fibril. Leading to the debundling of thick-fibrils into their constitutive thin-fibrils (C).</p

Type II collagen fibrils decomposed into their basic aggregates (viewed via X-ray diffraction and TEM).

<p>Some parts of the antibody treated samples maintain a loose alignment of the thin-fibrils allowing them to be analyzed with small angle X-ray diffraction (A), and insert B. An 11 and 4.5 nm packing function are apparent, which appear to correspond to the approximate diameter of the thin-fibrils (insert of C) and microfibrils (D). Native thick fibrils are shown in C as a comparison to the decomposition product (thin-fibrils).</p

TEM images of human articular cartilage preparations.

<p>A) Section of native human articular cartilage, incubated in TBS, that has collagen type II fibrils of regular 30–50 nm diameter (control for samples B–D). B) Section of human articular cartilage treated with ABC lyase for 24 h with some thin fibrils of collagen type II. C) Section of human articular cartilage treated with Guanidine hydrochloride for 24 h with presence of thin 10–15 nm fibrils and normal thick fibrils (fibril bundles). D) Section of human articular cartilage, treated with anti-biglycan antibody for 24 h, shows some collagen type II thin fibrils as well as fibrils of regular 30–50 nm diameter. Arrows point to decomposing fibrils.</p

Helical and non-helical organization of collagen.

<p>The non-helical, folded C-terminal end of the collagen molecule (top) extending from the triple-helical region (below). The electron density of neighboring collagen molecules can be seen along side the chain traced segment (red). The GPO₅ domain is indicated in white.</p

Patterson functions of collagen model structure factors 00L (meridional) series.

<p>A) Comparison of GPO (7/2 model) and GPO with collagen sequence threaded to check if amino acid sequence effects periodicities detected by the Patterson function. It does not appear so. B) As (A) except for GAA (10/3 model). C) Patterson functions of collagen types I and II are compared with those from the GAA and GPO coordinate models with the collagen sequence threaded onto them. The semi-transparent arrows mark: red, the maximum of the GAA (10/3) helix model pitch and repeat periods, the black arrows mark the collagen I and II respective positions for these periods. Note that the collagen experimental data show periods that are longer then the 7/2 and do seem to almost reach the 10/3 expected range. This could be interpreted to mean that both helical symmetries are found in native fibrillar collagen in addition to other possible conformations.</p

Patterson functions of the type I and II collagen 00L (meridional) series.

<p>A) Patterson function from 0.0–0.5D, the inverse (0.5–1.0) half of the Patterson function is not shown. The fractional distances between periodicities indicated in the functions has been multiplied by 67 nm (the length of the one dimensional unit cell – the D-period) for comparison with the helix symmetry periods. B) Enhanced view of the Patterson function range of interest for the helix symmetry periodicities. C) Table of key helix periodicities for comparison with A and B (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089519#pone-0089519-g004" target="_blank">Figure 4</a>).</p

Greatly simplified organizational hierarchy of fibrillar collagen structure (from polypeptide to fibril)

<p>A. The collagen-forming polypeptide chains contain a large helix-forming domain with the repeat amino acid sequence Gly-X-Y, where X and Y are occupied by Pro or Hyp more frequently than other residues, but only account for approximately 1/6 of the total amino acid content (see for instance human sequence: ExPASy sequence data bank codes; P02452 and P08123). An arrow points to the figure element that shows that three polypeptides form the collagen monomer. The large triple-helix (super-helix) domain of approximately 300 nm in length is flanked by non-helical telopeptides (N and C, shown). The 6–8.6 nm dimension indicates the repeat of the triple-helix (36; 37). B. Collagen molecules are staggered approximately 67 nm from one another in the formation of microfibril aggregates. The microfibrils are D-periodic (D = 67 nm), and in each D-period, two monomers coil, or partially coil, around each other giving the appearance of another helix-like feature in the structural hierarchy (3). C. Cross-sectional view of the collagen molecular packing of a type I collagen fibril (11). Each circle represents one collagen molecule in cross-section (at the axial level of 0.44D). at the 0.44 D position. Next to B to C arrow, cross-section of an isolated microfibril. D) Archival image (Orgel laboratory) of the wide angle fiber diffraction pattern of type I collagen from rat tail tendon. The distinctly different but superimposed non-crystalline and crystalline diffraction patterns are indicated. Previous fiber diffraction studies of collagen's helical structure have concentrated on the non-crystalline part of the pattern, in this present study, we analyze crystalline diffraction data.</p

Experimental setup.

<p>1064-nm laser beam 1 is generated by Nd:YAG laser 2, steered by galvo-mirrors 3 and directed by dichroic mirrors 4 into trapping objective 5. Trapping objective focuses laser beam inside sample chamber 6 to create optical trap. After passing through sample chamber, laser beam is collected by tracking objective 7 and projected by dichroic mirror 8 on GaAs quadrant photodiode 9 (for XY detection) and photodiode 10 (for Z detection) after splitting by splitter-cube 11. Experiments are visualized with CCD camera 12 with visible light 13 being generated by overhead LED 14. Top and bottom of the sample chamber are made of glass coverslips 15 and 16 with TBS buffer 17 in between. Collagen fiber bundle 18 with cantilever-like fiber 19 is placed on the bottom coverslip. Microsphere 20 is attached to the cantilever-like fiber with the optical trap 21 and then displaced perpendicularly to the fiber (in the direction shown by red arrow).</p

Force-displacement curve and its linear fit.

<p>Nominal strain (top axis) is calculated as a relation between fiber length and bead displacement. The fitted region (from 1% to 3–4% nominal strain) is shown in color. We repeat each bending experiment five times (different colors) to improve precession and check reproducibility. The average slope for five runs is 7.32·10⁻⁶ N/m and standard deviation is 11%. <i>x</i>_<i>i</i> = 28.3 μm.</p