Effect of 3-hydroxyproline residues on collagen stability

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.     

Der Einfluss von D-Aminosäureresten auf das konformative Verhalten von Kollagenmodellpeptiden

The influence of D -amino acid residues upon the conformative behaviour of collagen model peptides. (L -Pro-L -Pro-Gly)₁₀ and (L -Pro-D (L )-Pro-Gly)₁₀ with 10 percent D -Prolin residues have been synthesized by the Merrifield technique. Further Poly-(Gly-Pro-Pro) and Poly-(Ala-Gly-Pro) with various amounts of D -amino acid residues have been synthesized by polycondensation of the proper tripeptides. The conformational behaviour of all these polypeptides has been studied by measurements at varying temperatures of optical rotation, circular dichroism and molecular weight. The results show that limited amounts of D -amino acid residues do not impedecollagen-like triple helical conformations. The denaturation temperatures, however, are considerably lowered by D -amino acid residues. The implications of these findings for the understanding of the properties of partially racemized degraded gelatins with relatively low gel melting points are discussed.     

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well‐defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15‐mer vascular endothelial growth factor mimicking α‐helical peptide (QK_L10A) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK_L10A H_α chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high‐resolution QK_L10A conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding–unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding–unfolding picture for the small peptide QK_L10A compatible with the nucleation–propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein–protein interactions.     

Al2O3/SnO2 Co-Nanoparticle Modified Grafted Collagen for Improving Thermal Stability and Infrared Emissivity

Al2O3/SnO2 co-nanoparticles were prepared with a modified sol-gel technique followed by a thermal treatment process. With these co-nanoparticles the grafted collagen-Al2O3/SnO2 nanocomposites were obtained using a supersonic dispersion method. X-ray diffraction, FT-IR analysis, transmission electron microscopy, TGA/DTA and infrared emissivity test were performed to characterize the resulting nanoparticles and nanocomposites, respectively. The Al2O3/SnO2 co-nanoparticles showed a narrow distribution of size between 20-40 nm and could be uniformly absorbed on the tri-helix scaffolds of the grafted collagen without any aggregation. The nanocomposites possessed better thermal stability and substantially lower infrared emissivity than the grafted collagen and Al2O3/SnO2 co-nanoparticles with an increase of degradation temperature from 39 to 210 ℃ and a decrease of infrared emissivity from 0.850 of the grafted collagen and 0.708 of the Al2O3/SnO2 co-nanoparticles to 0.424, which provided a potential application of the nanocomposites to areas such as photoelectronics.     

Effect of gelatin on properties of <Emphasis Type="Italic">Luciola mingrelica</Emphasis> firefly luciferase

An ATP-reagent containing thermostable mutant of Luciola mingrelica firefly luciferase, MgSO₄, components of buffer solution, and stabilizers, which is widely used to detect nano- and picomolar concentrations of adenosine-5′-triphosphate (ATP) in various biological samples, is been object of this study. The activity, stability, and analytical characteristics of the ATP-reagent have been assessed in the presence and absence of 5% gelatin and in gelatin gel. The solution of ATP-reagent was obtained at 30°C and a gelatin concentration of 5%, while gel formation occurred at 22°C. The gelatin addition decreased the activity and stability of luciferase slightly. The sensitivity of ATP detection (above 0.96) did not depend on the gelatin presence and the aggregate state of the disperse system. Limits of detection were 2 × 10⁻¹², 7 × 10⁻¹³, and 7 × 10⁻¹⁴ M ATP, when the ATP-reagent was used in gelatin films and in the solution in the presence of 5% gelatin and in the absence, respectively. It was shown that the storage of ATP-reagent in gelatin gel not only preserved enzymatic activity, but protected the enzyme from bacterial contamination, which was the cause of the enzyme activity loss.     

Effects of hydroxy compounds on gel formation of gelatin

 Network formation of gelatin gel is known to consist of three-dimensionally cross-linked triple helices among polypeptide chains. The effects of added low molecular weight mono-ols, diols and polyols on the higher-order structure formation of gelatin chains were investigated using the following measurements: melting temperature, viscoelasticity and spin-lattice relaxation time (T ₁) of H¹⁷ ₂O of gels, and circular dichroism spectra of diluted gelatin solutions. Furthermore, hydration behaviors of these hydroxy compounds were evaluated from the dynamic hydration numbers (n _DHN) derived from T ₁ of H¹⁷ ₂O in the solutions. It was found that network structures of gelatin gels containing hydoxy compounds were influenced by the number and position of hydroxyl groups as well as the number of carbon atoms of these coexisting compounds. The effect of hydroxyl groups of hydroxy compounds was considered to stabilize the helices among gelatin chains. Especially, the addition of polyols with large number of hydroxyl groups increased the number of cross-linking junctions in the gel networks, which consist of the aggregation among the helices. On the contrary, the effect of carbon atoms of hydroxy compounds is to disturb the formation of the helices and the aggregation among the helices. Received: 18 April 1996/Accepted: 23 July 1996     

Study on the morphology, crystalline structure and thermal properties of yellow ginger starch acetates with different degrees of substitution

Yellow ginger starch acetates with different degrees of substitution (DS) were prepared by reacting native starch with glacial acetic acid/acetic anhydride using sulfuric acid as catalyst. X-ray diffraction (XRD) of acetylated starch revealed that the crystal structure of native starch was disappeared and new crystalline regions were formed. Their formation was confirmed by the presence of the carbonyl signal around 1750 cm⁻¹, as well as the reduced hydroxyl groups, in the Fourier transform infrared spectroscopy (FT-IR). The scanning electron microscopy (SEM) suggested most of the starch granules disintegrated with many visible fragments along with the increasing DS. The thermal behavior of the native starch and starch acetate were investigated using thermogravimetric analysis (TGA) and differential thermal analysis (DTA), it was observed that the thermal stability of acetylated starch depends on the degree of substitution. Thermal stability of high DS acetylated starch is much better than that of the original starch when DS reached to 2.67.     

Liesegang rings of lead iodide Part II

DistinctLiesegang rings of lead iodide in gelatin gel are not formed as the salt forms a very stabilised colloid in gelatin medium. The stability of the colloid has been investigated. This sol is precipitated by potassium iodide and barium nitrate. Well definedLiesegang rings of lead iodide are formed in the gelatin gel in the presence of electrolytic barium nitrate.     

Organogels from liquid and gaseous hydrocarbons

It is shown that n-butane, condensed in autoclave, can readily dissolve the anionic surfactant AOT (bis-2-ethylhexylsodium sulphosuccinate) and that this solution solubilizes water up tow ₀=molar ratio/water-AOT).The microemulsion can be transformed into a gel by solubilizing gelatin in the water microphase. These microemulsion gels are characterized by a very high viscosity (several hundred poise). However, the partial vapour pressure of the n-butane component in the gel is as high as in the liquid state. Infrared studies show that part of the water in the gel is still free.Stability diagrams of this system and¹H-NMR studies to assess the state of water are presented. Analogously to reverse micelles from liquid hydrocarbons, the chemical shift of water protons is shifted to higher fields with respect to bulk water. This effect increases with decreasing temperature andw ₀. At loww ₀ values (< 10), a part of the water in the micelles does not freeze even at –35°C.Due to the rapid evaporation of n-butane from the gel, gelatin films can be prepared simply by casting. Differential scanning calorimetry studies of the gels (from n-butane as well as from isooctane and other hydrocarbons) show that the films have a thermal stability comparable with, and sometimes higher than, gelatin films prepared by conventional methods. Preliminary permeability studies of these films with solutions of amino acid derivatives and with gas such as nitrogen and helium are presented.     

A rapid and efficient algorithm for packing polypeptide chains by energy minimization

An improved algorithm for packing polypeptide chains with fixed geometry, which converges to a local energy minimum rapidly and efficiently, is described. The speed of convergence of the new algorithm is comparable to that of existing algorithms for minimizing the energies of single polypeptide chains, and it is several times greater than the speed of convergence of previous algorithms for minimizing the energy of structures consisting of several polypeptide chains. The algorithm has been used to minimize the energy of three-stranded (L -Ala)₈ β-sheets, three-stranded (L -Val)₆ β-sheets, and five-stranded (L -Ile)₆ β-sheets, starting from regular structures found previously; of the three-stranded regular and truncated (Gly-L -Pro-L -Pro)₄ structures used in earlier work to model collagen; and of the stacked β-sheet (L -Ala-GLy)₆ structures used to model silk. The antiparallel L -Ala β-sheet, and Gly-Pro-Pro triple helices, and the silk II structure remained nearly regular after energy minimization, but by contrast with results from earlier computations the other structures became significantly irregular. © 1994 by John Wiley & Sons, Inc.     

Fe(III)-binding collagen mimetics

The synthesis and characterization of hydroxamic acid containing single-chain and TRIS-assembled (where TRIS is tris(carboxyethoxymethyl)aminomethane) collagen mimetics are reported. We have engineered an Fe(III)-binding domain by placing a hydroxamic acid group at the C termini of collagen mimetic chains composed of the Gly-Pro-NLeu sequence. The circular dichroism spectra and thermal denaturation studies show an enhancement in triple-helical thermal stability upon the addition of Fe(III) for the TRIS-assembled structure. No triple-helical structure was detected for the single-chain collagen mimetic. From the absorbance shown in the UV-vis spectra, we believe that the thermal stabilization of the triple helix is the direct result of a coordination complex between Fe(III) and the hydroxamate groups tethered to the C termini of the collagen mimetic peptide chains.     

Triple-Helix-Stabilizing Effects in Collagen Model Peptides Containing PPII-Helix-Preorganized Diproline Modules

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross-linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline-mimicking modules (ProMs), which were preorganized in a PPII-helix-type conformation by a functionalizable intrastrand C₂ bridge. Results of CD-based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main-chain preorganization, ring-flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.     

Thermosensitive gel formation of novel polypeptides containing a collagen‐derived Pro‐Hyp‐Gly sequence and an elastin‐derived Val‐Pro‐Gly‐Val‐Gly sequence

A triple‐helix‐forming collagen model peptide, (prolyl‐trans‐4‐hydroxyprolyl‐glycyl)₁₀ [(Pro‐Hyp‐Gly)₁₀], and a thermosensitive elastin‐derived pentapeptide, valyl‐prolyl‐glycyl‐valyl‐glycyl (Val‐Pro‐Gly‐Val‐Gly), were copolymerized in various mole ratios using 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride and 1‐hydroxybenzotriazole in dimethyl sulfoxide at 20 °C. All of the obtained polypeptides have molecular weight higher than 10³ and contain a triple‐helical structure, and showed an inverse phase transition from transparent solution to turbid suspension in response to a rise in temperature. The lower critical solution temperature of the polypeptide solution decreased upon increasing the content of Val‐Pro‐Gly‐Val‐Gly. Furthermore, polypeptides containing 82–86 mol % of Val‐Pro‐Gly‐Val‐Gly in composition showed reversible gel formation, suggesting that (Pro‐Hyp‐Gly)₁₀ acts as a hydrated unit and Val‐Pro‐Gly‐Val‐Gly acts as a thermosensitive crosslinking point. These biodegradable thermosensitive polypeptides may be useful for biomedical applications, including, as a scaffold for tissue regeneration. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 6048–6056, 2005     

Triple‐Helix‐Stabilizing Effects in Collagen Model Peptides Containing PPII‐Helix‐Preorganized Diproline Modules

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross‐linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline‐mimicking modules (ProMs), which were preorganized in a PPII‐helix‐type conformation by a functionalizable intrastrand C₂ bridge. Results of CD‐based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main‐chain preorganization, ring‐flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.     

Conformational structure of the tetrapeptide Boc–Aib–Leu–Leu–Aib–OMe–the central fragment of the nonapeptide antibiotic leucinostatin A

The conformational structure of the tetrapeptide Boc–Aib–Leu–Leu–Aib–OMe has been investigated by the PCILO method. The computational results show the formation of two closed β-turns, both of which are of type III, and the peptide backbone folds into a right-handed 3₁₀-helical conformation stabilized by two intramolecular 4 → 1 hydrogen bonds. The helix thus formed generates a pore of ～3 Å along helix axis with hydrophobic amino acid side chains located on the outside of the helix, and this tendency of leucine side chains may enable leucinostatin A to fit into the membrane bilayer. The pore thus formed is cation-selective, and through this pore, the cation can pass only in a single file.     

Stabilization of a β-Hairpin Conformation in a Cyclic Peptide Using the Templating Effect of a Heterochiral Diproline Unit

A straightforward and effective method of stabilizing a β-hairpin conformation in a cyclic protein loop mimetic is described, which exploits the templating effect of a heterochiral D -Pro-L -Pro dipeptide unit. A twelve-residue β-hairpin loop was grafted from the extracellular interferon γ receptor onto the heterochiral D -Pro-L -Pro dipeptide template to afford a fourteen-residue cyclic peptide. The residues directly attached to the D -Pro-L -Pro template are shown by NMR spectroscopy to structurally mimic corresponding residues in adjacent antiparallel β-strands in the receptor. MD Simulations with and without time-averaged distance restraints support this view and indicate that the tip of the loop is more flexible, as inferred also for the receptor protein from crystallographic data. The templating effect of the heterochiral diproline unit also promotes efficient backbone cyclization of the fourteen-residue linear peptide precursor, suggesting that a wide variety of related protein loop mimetics incorporating the D -Pro-L -Pro template might be readily accessible.     

Liesegang rings of copper chromate in gelatin gel

Summary DistinctLiesegang Rings of Copper chromate in gelatin gel are not formed as the salt forms a very stable colloid in this medium. As this sol is coagulated by KH₂PO₄, well defined rings of Copper chromate in gelatin gel are formed in the presence of this electrolyte.

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Zusammenfassung DeutlicheLiesegang-Ringe werden in Kupfer-Chromat und Gelatine-Gel nicht gebildet, da das Salz ein sehr stabiles Kolloid in diesem Medium darstellt. Wenn dieses Sol durch KH₂PO₄ koaguliert wird, entstehen definierte Ringe von Kupfer-Chromat im Gelatine-Gel bei Gegenwart dieses Elektrolyten.

     

The new science of protein mimetics

New chemistries have been developed for de novo protein design. Protein mimetics of different structural and functional properties such as synthetic peptide ligases and Dn symmetrical helical bundles have been reported. The Template-Assembled Synthetic Protein (TASP) method (as well as the ßMolecular Kit' approach) has also been utilized to prepare protein-like molecules. Here we report the synthesis of single chain, scaffold (TRIS)- and dendrimer-assembled collagen mimetics composed of the Gly-Nleu-Pro sequence where Nleu denotes N-isobutyl glycine. From the CD spectra and the thermal denaturation studies it can be seen that the collagen mimetics prepared form stable triple helices except the single chain structure. Furthermore, the 162-residue collagen mimetic dendrimer exhibits enhanced triple helical stability compared to the equivalent scaffold-terminated structure by an increase in the melting temperature in both H₂O and 2:1 ethylene glycol/H₂O (4°C and 12°C respectively). The concentration dependence for the melting transition of the collagen mimetic dendrimer was measured from which it was determined that the stabilization effect arises from the intramolecular clustering of the triple helical arrays about the core structure. This ensemble excludes solvent from the interior portion of the array which stabilizes the triple helix cluster.     

Evaluation of a novel nanocrystalline hydroxyapatite powder and a solid hydroxyapatite/Chitosan-Gelatin bioceramic for scaffold preparation used as a bone substitute material

Artificially fabricated hydroxyapatite (HAP) shows excellent biocompatibility with various kinds of cells and tissues which makes it an ideal candidate for a bone substitute material. In this study, hydroxyapatite nanoparticles have been prepared by using the wet chemical precipitation method using calcium nitrate tetra-hydrate [Ca(NO₃)₂.4H₂O] and di-ammonium hydrogen phosphate [(NH₄)₂ HPO₄] as precursors. The composite scaffolds have been prepared by a freeze-drying method with hydroxyapatite, chitosan, and gelatin which form a 3D network of interconnected pores. Glutaraldehyde solution has been used in the scaffolds to crosslink the amino groups (|NH₂) of gelatin with the aldehyde groups (|CHO) of chitosan. The X-ray diffraction (XRD) performed on different scaffolds indicates that the incorporation of a certain amount of hydroxyapatite has no influence on the chitosan/gelatin network and at the same time, the organic matrix does not affect the crystallinity of hydroxyapatite. Transmission electron microscope (TEM) images show the needle-like crystal structure of hydroxyapatite nanoparticle. Scanning Electron Microscope (SEM) analysis shows an interconnected porous network in the scaffold where HAP nanoparticles are found to be dispersed in the biopolymer matrix. Fourier transforms infrared spectroscopy (FTIR) confirms the presence of hydroxyl group (OH^-) , phosphate group (PO^3-₄) , carbonate group (CO^2-₃) , imine group (C=N), etc. TGA reveals the thermal stability of the scaffolds. The cytotoxicity of the scaffolds is examined qualitatively by VERO (animal cell) cell and quantitatively by MTTassay. The MTT-assay suggests keeping the weight percentage of glutaraldehyde solution lower than 0.2%. The result found from this study demonstrated that a proper bone replacing scaffold can be made up by controlling the amount of hydroxyapatite, gelatin, and chitosan which will be biocompatible, biodegradable, and biofriendly for any living organism.     

Synthetic thermally reversible gel systems. V

Differential thermal analysis has been used to study the fusion of aqueous thermally reversible gels of gelatin and polyacrylylglycinamide (PAG). In the case of gelatin gels, endotherms close to the melting point are readily observed and these are sometimes preceeded by a small exothermic heat of gel reorganization. Calculations are presented to show that breaking of the gelatin gel network requires only a small fraction of the observed endothermic heat of fusion and that most of the heat is required for melting larger crystallites within gelatin aggregates and for perhaps a helix → coil transition. Failure to observe endotherms by DTA over the known temperature range of fusion of PAG gels is consistent with prior measurements and conclusions. The noncrystallinity of PAG gels and soluble aggregates together with a heat of crosslinking of only ?5 to ?10 kcal/mole of crosslinks places the heat of fusion of PAG gels outside the lower limits of DTA sensitivity.