Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta.

Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains.     

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).     

Procollagen triple helix assembly: an unconventional chaperone-assisted folding paradigm

Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30-34 degrees C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50-200 microM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization.     

The peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 and acetyl-(Gly-Pro-3(S)Hyp)10-NH2 do not form a collagen triple helix

Hydroxylation of proline residues in the Yaa position of the Gly-Xaa-Yaa repeated sequence to 4(R)-hydroxyproline is essential for the formation of the collagen triple helix. A small number of 3(S)-hydroxyproline residues are present in most collagens in the Xaa position. Neither the structural nor a biological role is known for 3(S)-hydroxyproline. To characterize the structural role of 3(S)-hydroxyproline, the peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 was synthesized and analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and 1H nuclear magnetic resonance spectroscopy. At 4 degrees C in water the circular dichroism spectrum indicates that this peptide was in a polyproline-II-like secondary structure with a positive peak at 225 nm similar to Ac-(Gly-Pro-4(R)Hyp)10-NH2. The positive peak at 225 nm almost linearly decreases with increasing temperature to 95 degrees C without an obvious transition. Although the peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 forms a trimer at 10 degrees C, sedimentation equilibrium experiments indicate that Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 is a monomer in water at 7 degrees C. To study the role of 3(S)-hydroxyproline in the Yaa position, we synthesized Ac-(Gly-Pro-3(S)Hyp)10-NH2. This peptide also does not form a triple helix in water. 1H Nuclear magnetic resonance spectroscopy data (including line widths and nuclear Overhauser effects) are entirely consistent, with neither Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 nor Ac-(Gly-Pro-3(S)Hyp)10-NH2 forming a triple helix in water. Therefore 3(S)-hydroxyproline destabilizes the collagen triple helix in either position. In contrast, when 3(S)-hydroxyproline is inserted as a guest in the highly stable -Gly-Pro-4(R)Hyperepeated host sequence, Ac-(Gly-Pro-4(R)Hyp)3-Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms as stable a trimer (Tm=49.6 degrees C) as Ac-(Gly-Pro-4(R)Hyp)8-Gly-Gly-NH2 (Tm=48.9 degrees C). Given that Ac-(Gly-Pro-4(R)Hyp)3-Gly-4(R)Hyp-Pro-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms a triple helix nearly as stable as the above two peptides (Tm=45.0 degrees C) and the knowledge that Ac-(Gly-4(R)Hyp-Pro)10-NH2 does not form a triple helix, we conclude that the host environment dominates the structure of host-guest peptides and that these peptides are not necessarily accurate predictors of triple helical stability.     

Limited proteolysis of type I collagen at hyperreactive sites by class I and II Clostridium histolyticum collagenases: complementary digestion patterns

The initial proteolytic events in the hydrolysis of rat tendon type I collagen by the class I and II collagenases from Clostridium histolyticum have been investigated at 15 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(I) and alpha 2 chains, which migrate at different rates in the buffer system employed. Experiments with the class I collagenases indicate that the first cleavage occurs across all three chains of the triple helix close to the C-terminus to produce fragments whose alpha chains have molecular weights of approximately 88,000. The second cleavage occurs near the N-terminus to reduce the molecular weight of the alpha chains to 80,000. Initial proteolysis by the class II collagenases occurs across all three chains at a site in the interior of the collagen triple helix to give N- and C-terminal fragments with alpha-chain molecular weights of 35,000 and 62,000, respectively. The C-terminal fragment is subsequently cleaved to give fragments with alpha-chain molecular weights of 59,000. These results indicate that type I collagen is degraded at several hyperreactive sites by these enzymes. Thus, initial proteolysis by these bacterial collagenases occurs at specific sites, much like the mammalian collagenases. These results with the individual clostridial collagenases provide an explanation for earlier data which indicated that collagen is degraded sequentially from the ends by a crude clostridial collagenase preparation.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Characterization of triple-helical conformations and melting analyses of synthetic collagen-like peptides by reversed-phase HPLC

There is a confusion in the application of circular dichroism (CD) spectroscopy in analyzing collagen's structure for the overlapping of the spectral shapes and positions of the collagen triple helix and poly(proline-II)-like structure. The unique repetitive sequence of the collagen triple helix is susceptible to misalignment during the spontaneous assembly. Such misaligned structures are usually difficult to be characterized by CD or NMR spectroscopy. Here, RP-HPLC was developed as a conformational characterization technique for synthetic collagen-like peptides based on the different hydrophobicities exhibited by the triple-helical and unassembled peptides. RP-HPLC was also used to study thermal transitions and to measure melting point temperatures (Tm) of the collagen-like peptides.     

Thermal stability of myosin rod from various species

The radius of gyration and fraction helix as a function of temperature have been determined for myosin rod from four different species: rabbit, frog, scallop, and antarctic fish. Measurements from sodium dodecyl sulfate gel electrophoresis indicate that all particles have the same molecular weight (approximately 130K). All fragments are nearly 100% alpha-helical at low temperatures (0-5 degrees C). The melting profiles for each are qualitatively similar in shape, but their midpoints are shifted along the temperature axis in the following order: antarctic fish (Tm = 33 degrees C), scallop (Tm = 39 degrees C), frog (Tm = 45 degrees C), and rabbit (Tm = 49 degrees C). Corresponding radius of gyration vs temperature profiles for each species are shifted to lower temperatures (approximately 5-8 degrees C) with respect to the optical rotation melting curves. From plots of radius of gyration vs fraction helix, we find a marked drop in the radius of gyration (from 43 to approximately 34 nm) with less than a 5% decrease in fraction helix for rabbit, frog, and antarctic fish rods, whereas the radius of gyration of scallop rod never exceeds 34 nm. Results indicate hinging of the myosin rod of each species. The thermal stabilities of the myosin rods shift in parallel with the working temperature of their respective muscles.     

Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Carbamylation differentially alters type I collagen sensitivity to various collagenases.

Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-product of urea, on free amino groups of proteins. Post-translational modifications are known to induce alterations in structural and functional properties of proteins, thus disturbing protein-protein or cell-protein interactions. We report the impact of carbamylation on type I collagen sensitivity to enzymatic proteolysis. Type I collagen was extracted from rat tail tendons and carbamylated by incubation with 0.1 M potassium cyanate at 37 degrees C for 2, 6 or 24 h. Degradation assays revealed that carbamylated collagen exhibited a greater resistance to collagenases (i.e. bacterial collagenase, matrix metalloproteinase(MMP)-1, MMP-8 and MMP-13), together with an increased sensitivity to MMP-2. Evaluation of collagen triple helix conformation by polarimetry indicated that local destabilizations of triple helix structure related to carbamylation could be responsible for the observed differences in sensitivity. These results confirm the crucial role of triple helix integrity in the degradation of type I collagen by MMPs, and support the deleterious impact of post-translational modifications in vivo by altering the balanced remodeling of collagen within connective tissue.     

Proteolysis of procollagen I.

1. Digestion of procollagen I which trypsin, pepsin or pronase performed at 20 degrees C causes the release of acidic non-collagenous fragments and hydroxyproline-rich fraction. Enzymatic proteolysis performed at 41 degrees C (above the temperature of denaturation) results in degradation of procollagen I to low-molecular peptides. 2. The hydroxyproline-rich fraction obtained by limited proteolysis of procollagen I with pepsin (at 20 degrees C) contains a material corresponding to alpha and beta subunits of tropocollagen. Reduction of the hydroxyproline-rich fraction released by trypsin or pronase (at 20 degrees C) causes the appearance of polypeptides similar to pro-alpha subunits.     

Effect of alcohols on the collagen-phosphatidylcholine interaction

The interaction of phosphatidylcholine dispersions with acid soluble collagen separated from the skin of one month-old swine was studied to define the conditions facilitating the association of the collagen with lipids. When acid soluble collagen and phosphatidylcholine dispersions were incubated in 75 mM citrate buffer of pH 3.7 at 25 degrees C, the reisolated collagen fibrils did not contain appreciable amounts of phosphatidylcholine. However, the presence of n-propanol greatly promoted the retention of phosphatidylcholine, the amount of phosphatidylcholine associated being nearly 30% of collagen on a weight basis under optimal conditions. In contrast, methanol, ethanol, isopropanol, and n-butanol did not appreciably enhance the association of phosphatidylcholine with collagen. A limited inhibition of phosphatidylcholine retention was observed upon addition of sodium chloride to the propanol medium. The interaction of phosphatidylcholine with acid soluble collagen decreased sharply when temperature was increased above 30 degrees C; almost no phosphatidylcholine-collagen association occured at 40 degrees C. It appears that the enhanced association in the presence of n-propanol is due to a looseing of the collagen triple helix that exposes hydrophobic sites necessary for the interaction. However, the conversion of the triple helical structure to the random coil conformation by heating prevents the association of phosphatidylcholine with acid soluble collagen.     

The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13: SEQUENCE SPECIFICITY FOR BINDING AND CLEAVAGE*

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly⁷⁷⁵–Leu⁷⁷⁶ in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.     

Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.

A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.     

Hydroxylation-induced stabilization of the collagen triple helix. Further characterization of peptides with 4(R)-hydroxyproline in the Xaa position

4(R)-Hydroxyproline in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence of collagen plays a crucial role in the stability of the triple helix. Since the peptide (4(R)-Hyp-Pro-Gly)10 does not form a triple helix, it was generally believed that polypeptides with a -Gly-4(R)-Hyp-Yaa-repeated sequence do not form a triple helix. Recently, we found that acetyl-(Gly-4(R)-Hyp-Thr)10-NH2 forms a triple helix in aqueous solutions. To further study the role of 4(R)-hydroxyproline in the Xaa position, we made a series of acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptides where Yaa was alanine, serine, valine, and allo-threonine. We previously hypothesized that the hydroxyl group of threonine might form a hydrogen bond to the hydroxyl group of 4(R)hydroxyproline. In water, only the threonine- and the valine-containing peptides were triple helical. The remaining peptides did not form a triple helix in water. In 1,2- and in 1,3-propanediol at 4 degrees C, all the soluble peptides were triple helical. From the transition temperature of the triple helices, it was found that among the examined residues, threonine was the most stable residue in the acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptide. The transition temperatures of the valine- and allo-threonine-containing peptides were 10 degrees lower than those of the threonine peptide. Surprisingly, the serine-containing peptide was the least stable. These results indicate that the stability of these peptides depends on the presence of a methyl group as well as the hydroxyl group and that the stereo configuration of the two groups is essential for the stability. In the threonine peptide, we hypothesize that the methyl group shields the interchain hydrogen bond between the glycine and the Xaa residue from water and that the hydroxyl groups of threonine and 4(R)hydroxyproline can form direct or water-mediated hydrogen bonds.     

Decreased collagen stability as a response to the loss of structural integrity of thyroid cartilage

The thermal behavior, birefringence properties, and the biochemical composition of thyroid cartilage tissues have been studied. The hyaline cartilage, which was visualized as a quasi-isotropic medium, was composed of type II collagen, which did not denature at temperatures up to 100 degrees C. However, in hyaline cartilage digested by trypsin, the denaturation of collagen occured at 60 degrees C. Collagen fibers in the perichondrium were composed of type I and II collagen and formed a highly organized anisotropic structure (birefringence about 4.75 x 10(-3)) with a melting temperature of about 65 degrees C. The temperature of collagen denaturation in perichondrium in the whole system perichondrium-hyaline cartilage increased up to 75 degrees C, indicating the immobilization of perichondrium collagen by the extracellular matrix of the hyaline constituent.     

Temperature-dependent higher order structures of the (Pro-Pro-Gly)₁₀-modified dendrimer

Collagen is the most abundant protein in mammals and is widely used as a biomaterial for tissue engineering and drug delivery. We previously reported that dendrimers and linear polymers, modified with collagen model peptides (Pro‐Pro‐Gly)₅, form a collagen‐like triple‐helical structure; however, its triple helicity needs improvement. In this study, a collagen‐mimic dendrimer modified with the longer collagen model peptides, (Pro‐Pro‐Gly)₁₀, was synthesized and named PPG10‐den. Circular dichroism analysis shows that the efficiency of the triple helix formation in PPG10‐den was much improved over the original. The X‐ray diffraction analysis suggests that the higher order structure was similar to the collagen triple helix. The thermal stability of the triple helix in PPG10‐den was higher than in the PPG10 peptide itself and our previous collagen‐mimic polymers using (Pro‐Pro‐Gly)₅. Interestingly, PPG10‐den also assembled at low temperatures. Self‐assembled structures with spherical and rod‐like shapes were observed by transmission electron microscopy. Furthermore, a hydrogel of PPG10‐den was successfully prepared which exhibited the sol‐gel transition around 45°C. Therefore, the collagen‐mimic dendrimer is a potential temperature‐dependent biomaterial. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 270–277, 2011.     

Polyamine-linked oligonucleotides for DNA triple helix formation.

The concept of antigene therapy of disease is based on the ability of an oligonucleotide (the therapeutic agent) to bind to double-stranded genomic DNA (the target associated with the disease). Examples are herein given of the linkage of a series of polyamines to a 21-mer homopyrimidine oligonucleotide. These conjugated 21-mers can each form a triple helix with an appropriate double-stranded homopurine-homopyrimidine DNA according to Hoogsteen base-pairing rules. No triple helix was found when unmodified third strand was used at 10 mM sodium phosphate, pH 6.5, 100 mM sodium chloride solution. In contrast, the spermine-conjugated oligonucleotide had a melting temperature of 42 degrees C. According to the melting profile, the appended spermine moiety was found to affect the Tm only of the triple helix, but not of the subsequent melting of the underlying double helix. The Tm enhancing ability of the spermine-conjugate was found to be better than that of other polyamine-conjugates.     

Proteolytic degradation of cold-water fish gelatin solutions and gels

The stability of cold-water fish gelatin (FG), both in solution and in the gel phase, has been studied as function of both temperature and exposure towards novel proteases of marine origin. A 1% (w/v) FG solution was readily degraded by such proteases above 20 degrees C, which was expected since FG at this temperature is a random coil molecule lacking the protective triple helical structure found in collagen. The dynamic storage modulus for a 10% (w/v) FG gel increased monotonically at 4 degrees C. Ramping the temperature to 6, 8 or 10 degrees C led to a drastic reduction in G', but an apparent partial recovery of the network (increasing G') was observed with time at all temperatures. In the presence of proteases, a lower storage modulus was observed. At constant 4 degrees C, an apparent maximum value was reached after curing for 2h followed by a decrease in G' indicating protease activity. Ramping of temperature in the presence of proteases led to an even more drastic reduction in G' and no recovery of structure was observed with time. In this case, the overall rheological behaviour is a complex function of both thermal influence as well as proteolytic activity. In an endeavour to quantify the effect of the presence of proteolytic enzymes on the gelatin network, rheological investigation were undertaken where the dynamic storage moduli were recorded on different 10% (w/v) FG samples that had been acid hydrolysed to yield different average molecular weights. A significant reduction in storage modulus for average molecular weights below 50 kDa was found. This critical molecular weight most probably reflects the on-set of a regime where shorter chain lengths prevent percolation due to an increase in the loose end and sol fraction as well as a reduction in the average length of the pyrrolidine-rich regions reducing the number of possible junction zones.