Thermodynamic studies of the assembly in vitro of native collagen fibrils.
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ABSTRACT:
SUBMITTER: Cooper A
PROVIDER: S-EPMC1179199 | biostudies-other | 1970 Jul
REPOSITORIES: biostudies-other
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Project description:The yeast inheritable phenotype [URE3] is thought to result from conformational changes in the normally soluble and highly helical protein Ure2p. In vitro, the protein spontaneously forms long, straight, insoluble protein fibrils at neutral pH. Here we show that fibrils of intact Ure2p assembled in vitro do not possess the cross beta-structure of amyloid, but instead are formed by the polymerization of native-like helical subunits that retain the ability to bind substrate analogues. We further show that dissociation of the normally dimeric protein to its constituent monomers is a prerequisite for assembly into fibrils. By analysing the nature of early assembly intermediates, as well as fully assembled Ure2p fibrils using atomic force microscopy, and combining the results with experiments that probe the fidelity of the native fold in protein fibrils, we present a model for fibril formation, based on assembly of native-like monomers, driven by interactions between the N-terminal glutamine and asparagine-rich region and the C-terminal functional domain. The results provide a rationale for the effect of mutagenesis on prion formation and new insights into the mechanism by which this, and possibly other inheritable factors, can be propagated.
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Project description:It has been established that the enzyme susceptibility of collagen, the predominant load-bearing protein in vertebrates, is altered by applied tension. However, whether tensile force increases or decreases the susceptibility to enzyme is a matter of contention. It is critical to establish a definitive understanding of the direction and magnitude of the force versus catalysis rate (k C ) relationship if we are to properly interpret connective tissue development, growth, remodeling, repair, and degeneration. In this investigation, we examine collagen/enzyme mechanochemistry at the smallest scale structurally relevant to connective tissue: the native collagen fibril. A single-fibril mechanochemical erosion assay with nN force resolution was developed which permits detection of the loss of a few layers of monomer from the fibril surface. Native type I fibrils (bovine) held at three levels of tension were exposed to Clostridium histolyticum collagenase A. Fibrils held at zero-load failed rapidly and consistently (20 min) while fibrils at 1.8 pN/monomer failed more slowly (35-55 min). Strikingly, fibrils at 23.9 pN/monomer did not exhibit detectable degradation. The extracted force versus k C data were combined with previous single-molecule results to produce a "master curve" which suggests that collagen degradation is governed by an extremely sensitive mechanochemical switch.
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Project description:BACKGROUND:Collagen, the most abundant human protein, is the principal component of the extracellular matrix and plays important roles in maintaining tissue and organ integrity. Highly resistant to proteolysis, fibrillar collagen is degraded by specific matrix metalloproteases (MMPs). Degradation of fibrillar collagen underlies processes including tissue remodeling, wound healing, and cancer metastasis. However, the mechanism of native collagen fibril degradation remains poorly understood. RESULTS:Here we present the results of high-resolution tracking of individual MMPs degrading type I collagen fibrils. MMP1 exhibits cleavage-dependent biased and hindered diffusion but spends 90% ± 3% of the time in one of at least two distinct pause states. One class of exponentially distributed pauses (class I pauses) occurs randomly along the fibril, whereas a second class of pauses (class II pauses) exhibits multistep escape kinetics and occurs periodically at intervals of 1.3 ± 0.2 ?m and 1.5 ± 0.2 ?m along the fibril. After these class II pauses, MMP1 moved faster and farther in one direction along the fibril, indicative of biased motion associated with cleavage. Simulations indicate that 5% ± 2% of the class II pauses result in the initiation of processive collagen degradation, which continues for bursts of 15 ± 4 consecutive cleavage events. CONCLUSIONS:These findings provide a mechanistic paradigm for type I collagen degradation by MMP1 and establish a general approach to investigate MMP-fibrillar collagen interactions. More generally, this work demonstrates the fundamental role of enzyme-substrate interactions including binding and motion in determining the activity of an enzyme on an extended substrate.
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Project description:A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2--3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.
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Project description:In this manuscript we use a two-dimensional coarse-grained model to study how amyloid fibrils grow towards an equilibrium state where they coexist with proteins dissolved in a solution. Free-energies to dissociate proteins from fibrils are estimated from the residual concentration of dissolved proteins. Consistent with experiments, the concentration of proteins in solution affects the growth rate of fibrils but not their equilibrium state. Also, studies of the temperature dependence of the equilibrium state can be used to estimate thermodynamic quantities, e.g., heat capacity and entropy.
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Project description:During the maturation in vitro of reconstituted collagen fibrils prepared from rat skin, the mechanical and thermal stability of collagen increased and the pepsin-solubility decreased. At the same time a larger fraction of the pepsin-soluble collagen attained a lower molecular thermal stability that resulted in a biphasic thermal transition of the soluble collagen. Type-I collagen, with a similar biphasic thermal transition, was isolated from acid-insoluble rat skin collagen.
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Project description:Wound contraction is an ancient survival mechanism of vertebrates that results from tensile forces supporting wound closure. So far, tissue tension was attributed to cellular forces produced by tissue-resident (myo-)fibroblasts alone. However, difficulties in explaining pathological deviations from a successful healing path motivate the exploration of additional modulatory factors. Here, it is shown in a biomaterial-based in vitro wound healing model that the storage of tensile forces in the extracellular matrix has a significant, so-far neglected contribution to macroscopic tissue tension. In situ monitoring of tissue forces together with second harmonic imaging reveal that the appearance of collagen fibrils correlates with tissue contraction, indicating a mechanical contribution of tensioned collagen fibrils in the contraction process. As the re-establishment of tissue tension is key to successful wound healing, the findings are expected to advance the understanding of tissue healing but also underlying principles of misregulation and impaired functionality in scars and tissue contractures.
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Project description:Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)(10), as with l-phenylalanine and l-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a'-d' (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a' > 1b' > 1c' > 1d', supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a-d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC(50) potency: 1a/1b >> 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.
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Project description:Collagen fibrils represent a unique case of protein folding and self-association. We have recently successfully developed triple-helical peptides that can further self-assemble 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 well-known 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 self-assembly 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 self-associate 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.
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