Triple Helical Structure Of Collagen Research Articles

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis.

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782-785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray "closed" conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

In Situ Unilateral 1H-NMR Studies of the Interaction Between Lead White Pigments and Collagen-Based Binders

The lead white pigment, 2PbCO3·Pb(OH)2, is thought to play a critical role in the degradation of paint in illuminated manuscripts. Cracking, flaking, and separation of paint films containing lead white on parchment and other works of art have been reported, and have been attributed to the interaction of the pigment with the binding media. A previous study by Fourier transform infrared spectroscopy showed lead white to induce a change in the state of hydration of protein-based binders, though the mechanism of this process is still not well understood. In this work, we apply in situ 1H unilateral nuclear magnetic resonance (NMR) to explore the nature of the interaction between lead white and collagen-based binders while at the same time we evaluate the feasibility of applying unilateral 1H-NMR to assess the condition of the paint in medieval-illuminated manuscripts and to follow up the effectiveness of consolidation treatments. Carr, Purcell, Meiboom, and Gill (CPMG) measurements reveal that the addition of lead white to binders derived from collagen, such as bone, rabbit skin or fish glues, increases the T 2eff relaxation constant of these binders. This effect is more pronounced at low relative humidity. The increase in T 2eff suggests that the pigment induces a change in the protein that leads to the formation of more mobile structures, such as peptide fragments, or the partial unfolding of the rigid collagen triple helical structure to a more mobile random coil. The presence of large random coil domains in a collagen-based film has been associated with a lower mechanical strength of the film and therefore with its likeliness to flake.

Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were extracted from scale of spotted golden goatfish (Parupeneus heptacanthus) with the yields of 0.46% and 1.20% (based on dry weight basis), respectively. Both ASC and PSC were characterised as type I collagen, containing α1 and α2 chains. β and γ components were also found in both collagens. Based on FTIR spectra, the limited digestion by pepsin did not disrupt the triple helical structure of collagen. ASC and PSC contained glycine (336–340 residues/1000 residues) as the major amino acid and had imino acids of 186–189 residues/1000 residues. Maximal transition temperatures (Tmax) were 41.58 and 41.01°C for ASC and PSC, respectively. From zeta potential analysis, net charge of zero was found at pH 4.96 and 5.39 for ASC and PSC, respectively. Both collagens exhibited high solubility in acidic pH (2–4) and were soluble in the presence of NaCl at concentration up to 20 and 30g/l for ASC and PSC, respectively.

Isolation and characterisation of collagen extracted from the skin of striped catfish ( Pangasianodon hypophthalmus)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of striped catfish ( Pangasianodon hypophthalmus) were isolated and characterised. The yields of ASC and PSC were 5.1% and 7.7%, based on the wet weight of skin, respectively, with the accumulated yield of 12.8%. Both ASC and PSC comprising two different α-chains (α1 and α2) were characterised as type I and contained imino acid of 206 and 211 imino acid residues/1000 residues, respectively. Peptide maps of ASC and PSC hydrolysed by either lysyl endopeptidase or V8 protease were slightly different and totally differed from those of type I calf skin collagen, suggesting some differences in amino acid sequences and collagen structure. Fourier transform infrared (FTIR) spectra of both ASC and PSC were almost similar and pepsin hydrolysis had no marked effect on the triple-helical structure of collagen. Both ASC and PSC had the highest solubility at acidic pH. A loss in solubility was observed at a pH greater than 4 or when NaCl concentration was higher than 2% (w/v). T max of ASC and PSC were 39.3 and 39.6 °C, respectively, and shifted to a lower temperature when rehydrated with 0.05 M acetic acid. Zeta potential studies indicated that ASC and PSC exhibited a net zero charge at pH 4.72 and 5.43, respectively. Thus, ASC and PSC were slightly different in terms of composition and structure, leading to somewhat different properties.

Doxycycline Delivery From Collagen Matrices Crosslinked With Tannic Acid

Collagen-doxycycline matrices crosslinked with a natural polyphenol – tannic acid for the treatment of infected wounds were obtained by the freeze-drying of the corresponding gels. FT-IR spectra show that the triple helical structure of collagen is preserved in all the matrices, doxycycline produce collagen crosslinking and the degree of crosslinking increases with tannic acid concentration. Digestion of matrices using collagenase confirms the crosslinking effect of doxycycline and tannic acid and the increasing of the crosslinking degree with the amount of acid. The release of doxycycline from the matrices crosslinked with tannic acid is slower than that from the uncrossliked one and decreases with increasing of acid concentration, according to FT-IR and digestion results, and follows the power law model, with a release exponent of about 0.4, which indicates an anomalous transport. The matrices containing doxycycline, tannic acid and their combination do not develop gram-positive (Staphylococcus aureus) or gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, fungi or leavens.

Fibrillogenesis in continuously spun synthetic collagen fiber

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.

Gamma-Crosslinked Collagen Gel without Fibrils: Analysis of Structure and Heat Stability

This paper reports an analysis of the structure and heat stability of two different collagen gels: conventional collagen gel (neutral gel) and gel without collagen fibrils (acidic gel), previously reported. We performed differential scanning calorimetry (DSC), observations by scanning electron microscope (SEM), observations by atomic force microscope (AFM), and small angle X-ray scattering (SAXS). Collagen fibrils were clearly observed in the neutral gel but not in the acidic gel by both SEM and AFM. A clear endothermic peak was observed at 53-55 degrees Celsius, representing disassembly of collagens in collagen fibrils in the neutral gel but not in the acidic gel. Only a small broad endothermic peak, at 35-43 degrees Celsius, representing the deformation of the triple helical structure of collagen, was observed in the acidic gel. The SAXS pattern also suggested that the neutral gel had a more heterogeneous structure than the acidic gel. The experimental results described here are compatible with the model proposed in a previous paper, and indicate more clearly that the acidic gel has no collagen fibrils and has a different molecular assembly state of Type I collagen than the neutral gel.

Isolation and Characterisation of collagen from the skin of brownbanded bamboo shark ( Chiloscyllium punctatum)

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of brownbanded bamboo shark ( Chiloscyllium punctatum) were isolated and characterised. The yield of ASC and PSC were 9.38% and 8.86% (wet weight basis), respectively. Based on protein patterns and TOYOPEARL ® CM-650M column chromatography, both collagens contained α- and β-chains as their major components. These were characterised as type I collagen with the cross-link of α2-chain. As digested by V8-protease and lysyl endopeptidase, peptide maps of both ASC and PSC were similar, but differed from that of type I collagen from calf skin. Fourier transform infrared (FTIR) spectra of both collagens were similar and pepsin hydrolysis had no effect on triple-helical structure of collagen. Transition temperature ( T max) of ASC and PSC were 34.45 and 34.52 °C, respectively, as determined by differential scanning colorimetry (DSC). From zeta potential study, the isoelectric points of ASC and PSC were estimated to be 6.21 and 6.56, respectively. Therefore, the skin of brownbanded bamboo shark could serve as an alternative source of collagen for different applications.

Effect of Hydration on the Thermal Stability of the Collagen Triple Helical Structure

Effect of Hydration on the Thermal Stability of the Collagen Triple Helical Structure

Toughing It Out in Membranes

The formation of crosslinks in the triple-helical structure of collagen is crucial to its function. In the case of collagen IV, which provides structural integrity to the basement membrane of animal tissues, the chemical nature of the crosslinks has been difficult to pin down. Vanacore et al. (p. [

Identification of Specific Hemopexin-like Domain Residues That Facilitate Matrix Metalloproteinase Collagenolytic Activity

Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285-295, 302-316, and 437-457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.

Collagen-curcumin interaction — A physico-chemical study

Curcumin is a widely used therapeutic agent with a wide spectrum of biological and physiological applications like wound healing and interacts with the skin protein, collagen. This work reports the effect of curcumin on various physico-chemical properties of collagen. The results suggest that significant changes in viscosity and surface tension occur on collagen interacting with curcumin. Secondary structure analysis using circular dichroism shows that curcumin does not alter the triple helical structure of collagen. Increasing concentration of curcumin resulted in aggregation of the protein. Further, curcumin imparts high level of thermal stability to collagen with shrinkage temperature of collagen increasing from 60 to 90°C.

Electrospinning of Collagen Nanofiber Scaffolds from Benign Solvents

Nanofiber scaffolds of collagen have been fabricated via electrospinning using benign solvent systems as a replacement for 1,1,1,3,3,3 hexafluoro-2-propanol. Simple binary mixtures of phosphate-buffered saline and ethanol have been found to be highly effective for electrospinning. FTIR spectra suggest that the triple helical structure of collagen was conserved after dissolution and electrospinning. Crosslinking of the electrospun collagen scaffolds was achieved with standard methods.

Crystal Structure of Human Type III Collagen Gly991–Gly1032 Cystine Knot-containing Peptide Shows Both 7/2 and 10/3 Triple Helical Symmetries

Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)(n) or (Gly-Pro-Hyp)(n). To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly(991)-Gly(1032) structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

How stable is a collagen triple helix? An ab initio study on various collagen and β‐sheet forming sequences

Collagen forms the well characterized triple helical secondary structure, stabilized by interchain H-bonds. Here we have investigated the stability of fully optimized collagen triple helices and beta-pleated sheets by using first principles (ab initio and DFT) calculations so as to determine the secondary structure preference depending on the amino acid composition. Models composed of a total of 18 amino acid residues were studied at six different amino acid compositions: (i) L-alanine only, (ii) glycine only, (iii) L-alanines and glycine, (iv) L-alanines and D-alanine, (v) L-prolines with glycine, (vi) L-proline, L-hydroxyproline, and glycine. The last two, v and vi, were designed to mimic the core part of collagen. Furthermore, ii, iii, and iv model the binding and/or recognition sites of collagen. Finally, i models the G-->A replacement, rare in collagen. All calculated structures show great resemblance to those determined by X-ray crystallography. Calculated triple helix formation affinities correlate well with experimentally determined stabilities derived from melting point (T(m)) data of different collagen models. The stabilization energy of a collagen triple helical structure over that of a beta-pleated sheet is 2.1 kcal mol(-1) per triplet for the [(-Pro-Hyp-Gly-)(2)](3) collagen peptide. This changes to 4.8 kcal mol(-1) per triplet of destabilization energy for the [(-Ala-Ala-Gly-)(2)](3) sequence, known to be disfavored in collagen. The present study proves that by using first principles methods for calculating stabilities of supramolecular complexes, such as collagen and beta-pleated sheets, one can obtain stability data in full agreement with experimental observations, which envisage the applicability of QM in molecular design.

Effect of UV irradiation on the physicochemical properties of collagen stabilized using aldehydes

AbstractCollagen is an important biomaterial, finding immense application in the field of wound healing. In this study, effect of UV irradiation on aldehydes crosslinked collagen has been carried out. Aldehydes find predominant application in crosslinking of collagen for various end uses. The physical and optical properties of aldehydes crosslinked collagen affected by UV irradiation have been detailed. Viscosity measurements have shown that aldehydes crosslinked collagen has better stability against UV radiation than native collagen. Circular dichroic studies showed that prolonged exposure to UV radiation changes the triple helical structure of collagen into random coil conformation. The difference spectra for both emission and absorption show that formaldehyde brings about more stability to collagen against UV irradiation than gluteraldehyde. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 3642–3648, 2007

Differentiation of Secreted and Membrane-Type Matrix Metalloproteinase Activities Based on Substitutions and Interruptions of Triple-Helical Sequences

The turnover of the collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology and has been ascribed to a small number of proteases. Several members of the matrix metalloproteinase (MMPs) family possess collagenolytic activity, and the mechanisms by which these enzymes process triple helices are beginning to be unraveled. The present study has utilized two triple-helical sequences to compare the cleavage-site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly approximately Met-Arg-Gly), while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn approximately Phe-Arg-Gly). Both sequences were selectively cleaved by MMP-13 while in linear form, but neither proved to be selective within a triple helix. This suggests that the conformational presentation of substrate sequences to a MMP active site is critical for enzyme specificity, in that activities differ when sequences are presented from an unwound triple helix versus an independent single strand. Differences in specificity between secreted and membrane-type (MT) MMPs were also observed for both sequences, where MMP-2 and MT-MMPs showed an ability to hydrolyze a triple helix at an additional site (Gly-Gln bond). Interruption of the triple helix had different effects on secreted MMPs and MT-MMPs, because MT-MMPs could not hydrolyze the Asn-Phe bond but instead cleaved the triple helix closer to the C terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P1 subsite to be able to efficiently process a triple-helical molecule. Analysis of individual kinetic parameters and activation energies indicated different substrate preferences within secreted MMPs, because MMP-13 preferred the interrupted sequence, while MMP-8 showed little discrimination between non-interrupted and interrupted triple helices. On the basis of the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such differences may be significant for understanding MMP mechanisms of action and aid in the development of selective MMP inhibitors.

Lagochilascaris minor third-stage larvae secrete metalloproteases with specificity for fibrinogen and native collagen

Dissemination of parasitic infections depends on migration through tissues and evasion from both hemostatic processes and immune responses from hosts. Metalloproteases play major roles in these mechanisms of pathogen-host interactions and, thus, are considered drug targets. In this study, we characterized metalloprotease activities in excretory/secretory (ES) products from third stage larvae (L3) of the ascarid Lagochilascaris minor, the causative agent of lagochilascariosis, which demonstrates an impressive migrating capacity across host tissues, including bone. Gel enzymography showed that ES products of L3 display two major gelatinolytic activities. Optimal proteolytic activity was found to occur at neutral/alkaline pH and was associated with two L. minor-secreted metalloproteases of 59 (SM59 Lm) and 114 kDa (SM114 Lm). We next showed that ES products of L3 were able to hydrolyze fibrinogen and collagen I at neutral pH, but not BSA, in an extensive manner. Furthermore, we demonstrated that ES products of L3 mediate hydrolysis of the triple helical structure of collagen I fibers in mouse mesentery. These results suggest that ES proteases of L3 might facilitate both L. minor migration through host tissues by hydrolyzing collagens of the extracellular matrix and evasion from host hemostatic mechanisms by degrading fibrinogen.

Stabilization of type I collagen against collagenases (type I) and thermal degradation using iron complex

The widespread application of collagen as a biomaterial warrants research in understanding the stabilization of the same. In this study, interaction of iron–tetrakis (hydroxymethyl) phosphonium (THP) complex with type I collagen has been investigated. DSC and hydrothermal measurement studies reveal that the shrinkage temperature of iron–THP treated rat tail tendon (RTT) collagen is 33°C higher than that of native RTT collagen. Fe–THP complex also brings about high degree of enzymatic stability to type I collagen. The effect of Fe–THP on the conformation of collagen was studied using circular dichroism and it was found that no major alterations in the triple helical structure of collagen occur on treatment with Fe–THP. It is observed from viscosity experiment results that though Fe–THP complex is able to bring about long range ordering of collagen, as evident from the thermal and enzymatic stability imparted to collagen, this ordering does not lead to any aggregation of collagen. Since THPS is reducing in nature, it is expected to keep iron in the +2 state and if THP chelates to Fe(II), the hydrolytic behavior of iron can also be controlled.