Characterization of Type I Collagen Fibril Formation Using Thioflavin T Fluorescent Dye

Morimoto, Katsura; Kawabata, Kazuya; Kunii, Saori; Hamano, Kaori; Saito, Takuya; Tonomura, Ben’ichiro

doi:10.1093/jb/mvp025

Cited by 26 publications

(25 citation statements)

References 36 publications

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“…AHCol can be considered as an essentially monomeric collagen in which 58% of molecules are composed of two ␣1 chains and 32% of ␣2 chains, with few cross-linkages between ␣ chains. These results are consistent with our previous data on tuna AHCol (30,31).…”

Section: Discussionsupporting

confidence: 94%

“…CD Spectrum of ASCol, PHCol, and AHCol-The triple-helical structure of each of ASCol, PHCol, and AHCol (prepared as described above) was confirmed by CD spectra as reported (30,31). The far-UV CD spectrum of the collagen preparations was measured by scanning through the range of 200 -250 nm with a CD spectropolarimeter J-820 (JASCO, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 94%

“…In this study, we compared the structural features of two types of protease-hydrolyzed collagen molecules, PHCol and AHCol, obtained from limited hydrolysis by pepsin and actinidain, respectively, from chicken skinderived ASCol. These collagen preparations were chosen because PHCol and AHCol derived similarly from tuna skin show interesting structural differences (30,31). Full sequence information of tuna skin collagen is not available.…”

Section: Discussionmentioning

confidence: 99%

“…We previously found that actinidain-hydrolyzed collagen (AHCol) of tuna retained its triple-helical structure at pH 4.0 and showed the same thermal stability as that of ASCol, as judged by circular dichroism (CD) spectroscopy. AHCol, however, was found to be essentially monomeric with no cross-linkages, and therefore it provides a unique system for further characterization (30,31).…”

mentioning

confidence: 99%

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Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

Kunii

Morimoto

Nagai

et al. 2010

Journal of Biological Chemistry

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We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of ␣1 and ␣2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N-and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the ␣1 1031 . We demonstrated that a synthetic nonapeptide mimicking the ␣1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the ␣1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.Type I collagen is the most abundant protein in connective tissue of all vertebrates. It forms highly ordered fibrils that are generally thought to provide mechanical strength and regulate cell function (1-5). Collagen is formed from tightly interwoven heterotrimers of two ␣1 chains and one ␣2 chain in a triplehelical coiled-coil structure. The triple helix consisting of GlyXaa-Yaa repeats is ϳ1,000 amino acid residues in length and is resistant to proteolysis except for specific collagenases that can cleave the helix (6, 7). The high content of 4-hydroxyproline in the Yaa position stabilizes the triple-helical structure (8, 9).Observations of in vitro collagen fibril formation suggest that the collagen molecule has sufficient structural information to assemble spontaneously under suitable conditions. This structural information has been partially revealed by atomic force microscopy, electron microscopy, spectroscopic analysis, and x-ray diffraction (10 -15). From this information, it appears that the telopeptide regions play an important role in the rate of fibril formation and in azimuthal and lateral growth of fibrils. Indeed, fibril formation of pepsin-hydrolyzed collagen (PHCol) 2 that has been partially hydrolyzed at the N-and C-telopeptide domains progresses more slowly than that of acidsoluble collagen (ASCol) (16,17). The recognition and association of collagen N-and C-telopeptides have been partly demonstrated in vitro (18 -25). The results imply that the Nand C-telopeptide regions are required to "seed" the interaction between collagen molecules. It was also concluded that the triple-helical structure is essential for fibril formation (4, 26).Our experimental approach to understand the mechanism of fibril formation was to investigate type I collagen that had been partially hydro...

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Section: Discussionsupporting

confidence: 94%

Section: Methodsmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

Kunii

Morimoto

Nagai

et al. 2010

Journal of Biological Chemistry

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“…The observation that ThT binds to the aggregates of peptides with 8-and 9-residue interruptions, as well as the EFYFDLRLK peptide, is consistent with a recent publication indicating that ThT binds to both collagen fibrils and amyloid fibrils. 25 …”

Section: Aggregate Morphologymentioning

confidence: 99%

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

Hwang¹,

Thiagarajan²,

Parmar³

et al. 2010

Protein Science

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The standard collagen triple-helix requires a perfect (Gly-Xaa-Yaa) n sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1-and 4-residue interruptions showed a localized perturbation within the triple-helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly-Pro-Hyp) n peptide context. All peptides in this set show decreases in triple-helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5-residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple-helix peptides containing 8-and 9-residue interruptions exhibit a strong propensity for self-association to fibrous structures. In addition, a small peptide modeling only the 9-residue sequence within the interruption aggregates to form amyloid-like fibrils with antiparallel b-sheet structure. The 8-and 9-residue interruption sequences studied here are predicted to have significant cross-b aggregation potential, and a similar propensity is reported for 10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple-helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.

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Collagen Mimicry with a Short Collagen Model Peptide

Mukherjee,

Sundarapandian,

Ayyadurai

et al. 2023

Macromol. Rapid Commun.

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Mimicking triple helix and fibrillar network of collagen through collagen model peptide(CMP) with short GPO tripeptide repeats is a great challenge. Herein, a minimalistic CMP comprising only five GPO repeats [(GPO)5] is presented. This novel approach involves the fusion of ultrashort peptide with the synergetic power of π‐system and β‐sheet formation to short CMP (GPO)5. Accordingly, a hydrogel‐forming, fluorenylmethoxycarbonyl (Fmoc)‐functionalized ultrashort peptide (NFGAIL) is fused at the N‐terminus and phenylalanine at the C‐terminus of (GPO)5 (Fmoc‐NFGAIL‐(GPO)5‐F‐COOH, FmP‐5GPO). At room temperature, it forms a robust triple helix in aqueous buffer solution and has a relatively high melting point of 35 °C. The fluorenyl motif stabilizes the triple helix by aromatic π–π interactions as in its absence, triple helix is not formed. NFGAIL, which forms a β‐sheet, also aids in triple helix stabilization via intermolecular hydrogen bonding and hydrophobic interactions. FmP‐5GPO forms highly entangled nanofibrils with a micrometer length, which have excellent cell viability. The achievement of stable triple helix and fibrils in such a short CMP(FmP‐5GPO) sequence is a challenging feat, and its significance in CMP‐based biomaterials is undeniable. The present strategy highlights the potential for developing new CMP sequences through intelligent tuning of fusion peptides and GPO repeats.

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Characterization of Type I Collagen Fibril Formation Using Thioflavin T Fluorescent Dye

Cited by 26 publications

References 36 publications

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

Collagen Mimicry with a Short Collagen Model Peptide

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