The collagen type I segment long spacing (SLS) and fibrillar forms: Formation by ATP and sulphonated diazo dyes |
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| Institution: | 1. Institute of Zoology, University of Mainz, 55099 Mainz, Germany;2. Institute for Cell and Molecular Biosciences, University of Newcastle, Newcastle-Upon-Tyne NE2 4HH, UK;1. Department of Zoology, University of Kalyani, Kalyani, Nadia 741325, India;2. Centre de Rechercheen Infectiologie, Centre de Recherche du CHU de Québec, University of Laval, Quebec City, Quebec, Canada;3. CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, India;1. Centro Chirurgico Toscano, Arezzo, Italy;2. Department of Urology, Humanitas Clinical and Research Centre, Humanitas University, Rozzano (Milan), Italy;1. C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV, USA;2. Department of Surgery, West Virginia University, Morgantown, WV, USA;3. Center for BioMedical Computing, University of Verona, Istituti Biologici, Blocco A, Strada le Grazie 8, 37134 Verona, Italy;1. Neurosciences Division—Human Genetics, CHUQ Research Center—CHUL, Quebec, QC, Canada;1. Axe Neurosciences, Research Center of the CHU de Quebec - CHUL, Québec, QC G1V 4G2, Canada |
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| Abstract: | The collagen type I segment long spacing (SLS) crystallite is a well-ordered rod-like molecular aggregate, ∼300 nm in length, which is produced in vitro under mildly acidic conditions (pH 2.5–3.5) in the presence of 1 mM ATP. The formation of the SLS crystallite amplifies the inherent linear structural features of individual collagen heterotrimers, due to the punctate linear distribution and summation of the bulkier amino acid side chains along the length of individual collagen heterotrimers. This can be correlated structurally with the 67 nm D-banded collagen fibril that is found in vivo, and formed in vitro. Although first described many years ago, the range of conditions required for ATP-induced SLS crystallite formation from acid-soluble collagen have not been explored extensively. Consequently, we have addressed biochemical parameters such as the ATP concentration, pH, speed of formation and stability so as to provide a more complete structural understanding of the SLS crystallite. Treatment of collagen type I with 1 mM ATP at neutral and higher pH (6.0–9.0) also induced the formation of D-banded fibrils. Contrary to previous studies, we have shown that the polysulphonated diazo dyes Direct red (Sirius red) and Evans blue, but not Congo red and Methyl blue, can also induce the formation of SLS-like aggregates of collagen, but under markedly different ionic conditions to those employed in the presence of ATP. Specifically, pre-formed D-banded collagen fibrils, prepared in a higher than the usual physiological NaCl concentration (e.g. 500 mM NaCl, 20 mM Tris-HCl pH7.4 or x3 PBS), readily form SLS aggregates when treated with 0.1 mM Direct red and Evans blue, but this did not occur at lower NaCl concentrations. These new data are discussed in relation to the anion (Cl−) and polyanion (phosphate and sulphonate) binding by the collagen heterotrimer and their likely role in collagen fibrillogenesis and SLS formation. |
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| Keywords: | Collagen Segment long spacing (SLS) crystallite Fibril D-Banding Diazo dye Sodium tri(poly)phosphate Negative staining |
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