Abstract
In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to "code" for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.
Highlights
Functional collagen fibrils are formed through the self-assembly of collagen triple helices
Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils
Each of the three pseudo-identical sequence units of Col108 contains a Col domain: a 108-residue segment consisting of residues 242–256, 296 –322, 434 – 478, and 515–535 of the ␣1 chain of human type I collagen, and an N-terminal (Gly-Pro-Pro)4 repeating sequence for added conformational rigidity; the U2 unit has two additional Gly-X-Y triplets flanking the Col domain introduced by the restriction enzyme sites used for the synthesis of the Col108 gene
Summary
Functional collagen fibrils are formed through the self-assembly of collagen triple helices. The banding pattern is characterized by an axially repeating feature of ϳ35 nm as observed by transmission electron microscopy and atomic force microscopy Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils. The fibrillogenesis involves the lateral association of collagen triple helices, the structural unit of collagen, to form long, smooth fibrils with a characteristic 67-nm axial structural feature known as the D-periodicity.
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