The thermal stability of triple helical structure plays a critical role in collagen biosynthesis, function and degradation. CD technique was utilized to characterize the thermal stability of synthetic collagen mimic peptides. Fluorescence spectroscopy is widely used with easy access all around the world because of its inexpensive instrumentation, low operation cost, easy operation, and high sensitivity. Here we have developed an alternative fluorescence method to detect the thermal stability of collagen mimic peptides. We have demonstrated that fluorescence spectroscopy could measure the thermal stability of collagen mimic peptides with low concentrations under different circumstances. This highly sensitive fluorescence self-quenching assay will greatly expedite the studies of sequence-dependent properties of collagen mimic peptides, and it has great potential in the application of determining the thermal stability of triple helix systems such as collagens, collectins, adiponectin, macrophage scavenger and C1q.Download high-res image (55KB)Download full-size imageA highly sensitive fluorescence self-quenching assay has been developed to detect the thermal stability of collagen mimic peptides under different circumstances. This assay will greatly expedite the studies of sequence-dependent properties of collagen mimic peptides.

We herein report the construction of a novel single stranded fluorescent collagen mimetic peptide by introducing a bulky FAM dye in the central region rather than the N terminus. Without the need for any prior thermal or ultraviolet treatment, the peptide probe can be conveniently applied to specifically target collagen in connective tissues for fluorescence imaging.

The synthesis of hematite mesocrystals with a tunable hierarchical nanostructure plays a critical role in the construction of improved functional materials. We have demonstrated that two recombinant collagen proteins can be used as superior biotemplates to produce hematite mesocrystals with easily tunable hierarchical nanostructures under hydrothermal conditions. Compared with previously reported proteins, collagen is able to regulate the hierarchical structures of hematite mesocrystals with a much lower concentration, and collagen can produce a richer diversity of hierarchical nanostructures, adding two novel diamond-like and sphere-like structures in addition to the three previously reported structures (spindle-like, olive-like, and ellipsoidal-like). The distinct (Gly–X–Y)n amino acid sequence pattern and triple helix structure may provide unique capability for collagen in protein-templated biomineralization. Our studies have indicated that sequence and structural differences in proteins may lead to a plethora of novel nanostructures, which would significantly contribute to the development of improved inorganic nanomaterials.

The construction of simple and efficient assays to detect collagen biomarkers plays a critical role in developing novel diagnosis and therapies for the highly prevalent chronic fibroproliferative diseases. Inspired by the successful development of various GO-based biosensors for DNA utilizing its well-known double helix structure, we have for the first time created a highly specific GO platform for sensing the collagen triple helix. We have designed a dye-labeled single stranded collagen (ssCOL) peptide probe to target a complementary single stranded collagen peptide sequence. We have revealed that GO binds with the ssCOL probe and quenches the fluorescence of the dye, while the hybridization of the ssCOL probe with its target collagen peptide GPO results in the retention of the fluorescence of the probe. We have demonstrated that this design provides a fluorescence-enhanced assay that is sensitive and selective to the target collagen peptide with little interferences from other proteins, and it can be applied for quantitative detection in complex biological fluids. These results indicate that this GO-based ssCOL platform has great potential in molecular diagnostics of fibroproliferative diseases. It may provide a novel strategy to construct efficient assays for the proteins containing triple helix motifs such as collectins, adiponectin, macrophage scavenger and C1q.

We have demonstrated that the incorporation of a dye-labeled collagen-like peptide in the homotrimeric versus heterotrimeric context results in visible color changes and distinct fluorescence. The unique fluorescence self-quenching assay can unambiguously determine the helix composition of heterotrimers at the nM level, far extending our capability to characterize a collagen triple helix.

•A specific FRET sensor is developed for detection of unfolded collagen fragments.•The sensor is based on the complex of GO and a designed collagen mimic peptide.•The complex prefers the collagen targets with complementary GPO-rich sequences.The unstructured collagen species plays a critical role in a variety of important biological processes as well as pathological conditions. In order to develop novel diagnosis and therapies for collagen-related diseases, it is essential to construct simple and efficient methods to detect unfolded collagen fragments. We therefore have designed a FITC-labeled collagen mimic triple helical peptide, whose adsorption on the surface of GO effectively quenches its fluorescence. The newly constructed GO/FITC–GPO complex specifically detects unstructured collagen fragments, but not fully folded triple helix species. The detection shows a clear preference for the collagen targets with complementary GPO-rich sequences. The conformation-sensitive, sequence-specific GO-based approach can be applied as an efficient biosensor for rapid detection of unfolded collagen fragments at nM level, and may have great potential in drug screening for inhibitors of unfolded collagen. It may provide a prototype to develop GO-based assays to detect other important unstructured proteins involved in diseases.

Natural interruptions in the repeating (Gly-X-Y)n amino acid sequence pattern are found normally in triple helix domains of all nonfibrillar collagens, while any Gly substitution in fibrillar collagens leads to pathological conditions. As revealed by our sequence analysis, two peptides, one modeling a natural G5G interruption (POALO) and the other one mimicking a pathological Gly-to-Ala substitution (LOAPO), are designed. Circular dichroism (CD), NMR, and computational simulation studies have discovered significant differences in stability, conformation, and folding between the two peptides. Compared with the Gly substitution sequence, the natural interruption maintains higher stability, higher triple helix content, and a higher folding rate while introducing more alterations in local triple helical conformation in terms of dihedral angles and hydrogen bonding. The conserved hydrophobic residues at the specific sites of interruptions may provide functional constraints for higher-order assembly as well as biomolecular interactions. These results suggest a molecular basis of different biological roles of natural interruptions and Gly substitutions and may guide the design of collagen mimic peptides containing functional natural interruptions.

The synthesis of hematite mesocrystals with a tunable hierarchical nanostructure plays a critical role in the construction of improved functional materials. We have demonstrated that two recombinant collagen proteins can be used as superior biotemplates to produce hematite mesocrystals with easily tunable hierarchical nanostructures under hydrothermal conditions. Compared with previously reported proteins, collagen is able to regulate the hierarchical structures of hematite mesocrystals with a much lower concentration, and collagen can produce a richer diversity of hierarchical nanostructures, adding two novel diamond-like and sphere-like structures in addition to the three previously reported structures (spindle-like, olive-like, and ellipsoidal-like). The distinct (Gly–X–Y)n amino acid sequence pattern and triple helix structure may provide unique capability for collagen in protein-templated biomineralization. Our studies have indicated that sequence and structural differences in proteins may lead to a plethora of novel nanostructures, which would significantly contribute to the development of improved inorganic nanomaterials.

In order to develop improved diagnosis and therapies for the highly prevalent chronic fibroproliferative diseases, it is of utmost importance to construct efficient assays to detect collagen biomarkers. We have successfully created a paper-based FRET assay for the fast, inexpensive and direct detection of collagen triple helix. We have demonstrated that the adsorption of the dye-labeled probe peptide onto the GO-immobilized paper quenches the fluorescence of the dye, while the hybridization of the probe peptide with the target collagen peptide results in the desorption of the probe peptide from GO, thus restoring the fluorescence. We have also shown that this novel assay is highly specific to the target collagen peptide sequence with little interference from other proteins, and it can be applied for quantitative detection in complex biological fluids. Our integration of the GO-based FRET assay with a patterned paper provides a powerful new tool for the detection of collagen molecules with many superior features: tiny volumes of samples, multichannel detection mode, easy operation and low-cost equipment. This assay may have promising applications to fast screen multiple target collagen sequences for the discovery of new collagen biomarkers.

The construction of simple and efficient assays to detect collagen biomarkers plays a critical role in developing novel diagnosis and therapies for the highly prevalent chronic fibroproliferative diseases. Inspired by the successful development of various GO-based biosensors for DNA utilizing its well-known double helix structure, we have for the first time created a highly specific GO platform for sensing the collagen triple helix. We have designed a dye-labeled single stranded collagen (ssCOL) peptide probe to target a complementary single stranded collagen peptide sequence. We have revealed that GO binds with the ssCOL probe and quenches the fluorescence of the dye, while the hybridization of the ssCOL probe with its target collagen peptide GPO results in the retention of the fluorescence of the probe. We have demonstrated that this design provides a fluorescence-enhanced assay that is sensitive and selective to the target collagen peptide with little interferences from other proteins, and it can be applied for quantitative detection in complex biological fluids. These results indicate that this GO-based ssCOL platform has great potential in molecular diagnostics of fibroproliferative diseases. It may provide a novel strategy to construct efficient assays for the proteins containing triple helix motifs such as collectins, adiponectin, macrophage scavenger and C1q.

We have demonstrated that the incorporation of a dye-labeled collagen-like peptide in the homotrimeric versus heterotrimeric context results in visible color changes and distinct fluorescence. The unique fluorescence self-quenching assay can unambiguously determine the helix composition of heterotrimers at the nM level, far extending our capability to characterize a collagen triple helix.