Chain conformation in the collagen molecule.

Quantitative X-ray diffraction data have been collected from stretched kangaroo tail tendon and used to test models for the conformation of the polypeptide chains in the collagen molecule. The magnitude of the unit twist of the molecular helix was estimated to be 107.1 ° ± 0.6 °, which is close to the value expected for a helix with ten units in three turns. The intensity data were used to carry out a linked-atom least-squares refinement of models based on two possible interchain hydrogen bonding schemes suggested by Rich &; Crick (1955, 1961). No stereochemically acceptable solution could be found for the hydrogen bonding scheme of model I, but a stereochemically satisfactory solution was found for the scheme of model II which gave a crystallographic R factor of 0.272.     

Variable chain conformations of renatured β‐glucan in dimethylsulfoxide/water mixture

We have firstly demonstrated the renaturation process of dissociated single chains of lentinan (s‐LNT) and the variable conformations of the renatured LNT (r‐LNT). The results from ultrasensitive differential scanning calorimetry and circular dichroism revealed that the variable structures including perfect triple helix, defective triple helix containing duplex segment, and single chains occurred in the renaturation of s‐LNT, depending on the renaturation time, solvent composition, molecular weight, and the mode of renaturation. When water was added into s‐LNT/dimethylsulfoxide (DMSO) to reach 95% (v/v), the classic low‐temperature intra‐triple‐helical conformational transition at ～10^°C (T₁) appeared within 4 h, indicative of a rapid reconstruction of triple helical structure. Besides, one newly endothermic peak at ～43^°C (T₂) simultaneously occurred, which was first ascribed to the melting of duplex segment in the imperfect triplex. The duplex stretches disappeared when DMSO reached 50%, in which single chains coexisted with triplex. Moreover, the duplex segment disappeared by slowly dropping water into s‐LNT/DMSO. This work suggested that the structure of r‐LNT could be controllable, and provided important information for their successful development and application in polymer and life science. © 2012 Wiley Periodicals, Inc. Biopolymers 97:988–997, 2012.     

Crystal structure of (Gly-Pro-Hyp)(9) : implications for the collagen molecular model

Collagens have long been believed to adopt a triple‐stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X‐ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single‐crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly‐Pro‐Hyp)₉ peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly‐Pro‐Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly‐Pro‐Hyp)₉ peptide adopts a triple‐stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 607–616, 2012.     

Structural hierarchy controls deformation behavior of collagen

The structure of collagen, the most abundant protein in mammals, consists of a triple helix composed of three helical polypeptide chains. The deformation behavior of collagen is governed by molecular mechanisms that involve the interaction between different helical hierarchies found in collagen. Here, we report results of Steered Molecular Dynamics study of the full-length collagen molecule (～290 nm). The collagen molecule is extended at various pulling rates ranging from 0.00003/ps to 0.012/ps. These simulations reveal a new level of hierarchy exhibited by collagen: helicity of the triple chain. This level of hierarchy is apparent at the 290 nm length and cannot be observed in the 7-9 nm models often described to evaluate collagen mechanics. The deformation mechanisms in collagen are governed by all three levels of hierarchy, helicity of single chain (level-1), helical triple helix (level-2), and hereby described helicity of the triple chain (level-3). The mechanics resulting from the three levels is described by an interlocking gear analogy. In addition, remarkably, the full-length collagen does not show much unwinding of triple helix unlike that exhibited by short collagen models. Further, the full-length collagen does not show significant unwinding of the triple helix, unlike that exhibited by short collagen. Also reported is that the interchain hydrogen bond energy in the full-length collagen is significantly smaller than the overall interchain nonbonded interaction energies, suggesting that the nonbonded interactions have far more important role than hydrogen bonds in the mechanics of collagen. However, hydrogen bonding is essential for the triple helical conformation of the collagen. Hence, although mechanics of collagen is controlled by nonbonded interchain interaction energies, the confirmation of collagen is attributed to the interchain hydrogen bonding.     

Conformational change of the triple-helical structure. II. Conformation of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n (Ala-Pro-Gly)m(Pro-Pro-Pro-Gly)n in an aqueous solution

Measurements of the molecular weight of (Pro-Pro-Gly)_n and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n, which were synthesized by the solid-phase method, revealed that they formed a trimer in an aqueous solution, and dissociated into single-stranded chains on warming. Accompanying the transition, a large decrease of optical rotation was observed, like the collagen–gelatin transition. The shape of the trimeric molecule was rodlike, and the dimensions were 12 Å in diameter and 2.8 Å per residue in length, regardless of the length of Ala-Pro-Gly sequences in a peptide chain. The data indicate that both Pro-Pro-Gly sequences and Ala-Pro-Gly sequences from the triple-helical structure similar to that of collagen in aqueous solution. All optical rotational dispersion (ORD) curves of solutions of the peptides were represented by a single-term Drude equation, and the Drude constant λ_c was 200 nm for all peptides regardless of the length of Ala-Pro-Gly sequences. The resemblance between the helical structure formed by Pro-Pro-Gly sequences and that by Ala-Pro-Gly sequences was also suggested by the formation of the hybrid triple helix from two kinds of peptide chains with different lengths of Ala-Pro-Gly sequences.     

Crystal and molecular structure of the amylose–DMSO complex

The crystal and molecular structure of the complex of amylose with dimethyl sulfoxide has been studied by a combination of stereochemical analysis, potential energy, and X-ray diffraction methods. The complex crystallizes in a pseudotetragonal unit cell with a = b = 19.17 Å and c (fiber axis) = 24.39 Å, with two antiparallel chains per unit cell and space group P2₁2₁2₁. The amylose chain is a left-handed 6₁(1.355) helix with three turns per crystallographic repeat. The O(6) rotational position is approximately gt. Dimethyl sulfoxide is located inside the helix with one DMSO molecule for every three glucose residues. An additional four DMSO molecules and eight water molecules each are located in the large interstices between chains, and it is the interaction of these molecules with the helix that results in the pseudotetragonal chain packing. The interstitial DMSO is the source of the previously reported additional layer lines, which are not consistent with the 8.13-Å amylose repeat distance. The final R factor for the layers with amylose contribution to the structure factors was 0.29, while the overall R factor was 0.35. The stereochemical packing analysis provided suitable phasing models for the subsequent X-ray refinement.     

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the α1, α2, and α3 chains of NC2, a stable heterotrimer with a disulfide bridge between α1 and α3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.     

Structure of the crystalline bilayer in the subgel phase of dipalmitoylphosphatidylglycerol

The structure of the subgel phase of dipalmitoylphosphatidylglycerol (DPPG) has been analyzed by X-ray diffraction techniques. Diffraction recorded from highly oriented DPPG specimens in the subgel phase extends to 2-A resolution. There are sharp lamellar reflections on the meridian, and other reflections lie on a series of wide-angle lattice lines parallel to the meridian and crossing the equator in the range of 8-2 A. The wide-angle lattice lines consist of radially sharp reflections centered on the equator of the X-ray film and also a series of broader, off-equatorial maxima. The lattice lines indicate that the DPPG molecules in each bilayer crystallize in a two-dimensional oblique lattice with dimensions a = 5.50 A, b = 7.96 A, and gamma = 100.5 degrees. These oblique lattices are not regularly aligned from bilayer to bilayer. Analysis of the lamellar diffraction shows that the bilayer has about the same thickness in the subgel and gel (L beta') phases. In the direction normal to the hydrocarbon chains, the chains are significantly closer together in the subgel phase as compared to the normal L beta' gel phase but have about the same separation as the chains in polyethylene and the crystalline n-alkanes. The bilayer thickness, area per lipid molecule, and intensity distribution along the lattice lines all indicate that in the subgel phase the hydrocarbon chains are tilted between 30 and 35 degrees from the normal to the bilayer plane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of water on the mechanical properties of gelatin films

The mechanical properties of gelatin films were studied in relation to the effect of water, and compared with those of collagen films. The S-shaped sorption isotherm was separated into an adsorption curve C₁ and dissolution curve C₂. From the C₂ curve, the interaction parameter χ₁ of Flory–Huggins' equation was calculated. The χ₁ of gelatin were larger than those of collagen at low relative humidities (RH), while they coincided with each other at high RH. It was found that a composite curve was made by shifting stress relaxation curves obtained at different humidities along the log time axis. The shift factor for the formation of the composite curve was analyzed by Fujita–Kishimoto's equation, which was based on the free volume theory. The parameter β, which expressed the extent of the contribution of sorbed water to the increase in the free volume of the system, was 0.05 in the range of C₂ from 0 to 0.08 (0–65% RH). This value was much smaller than 0.16 for collagen. The value was 0.16 in the range of C₂ higher than 0.08, which was equal to that of the collagen. Dynamic shear modulus G′, loss modulus G″, and tan δ were determined as functions of RH. The gelatin film extended more than 100% at 73% RH under the very small stress of about 10⁷ dyn/cm². This corresponds to the region where β changes from 0.05 to 0.16, although such a phenomenon was not observed in the collagen film. The wide-angle X-ray pattern of extended gelatin was similar to that of renatured collagen fiber.     

Effect of aqueous ethanol on the triple helical structure of collagen

Collagen, the most abundant protein in mammals, is widely used for making biomaterials. Recently, organic solvents have been used to fabricate collagen-based biomaterials for biological applications. It is therefore necessary to understand the behavior of collagen in the presence of organic solvents at low (≤50 %, v/v) and high (≥90 %, v/v) concentrations. This study was conducted to examine how collagen reacts when exposed to low and high concentrations of ethanol, one of the solvents used to make collagen-based biomaterials. Solubility testing indicated that collagen remains in solution at low concentrations (≤50 %, v/v) of ethanol but precipitates (gel-like) thereafter, irrespective of the method of addition of ethanol (single shot or gradual addition); this behavior is different from that observed recently with acetonitrile. Collagen retains its triple helix in the presence of ethanol but becomes thermodynamically unstable, with substantially reduced melting temperature, with increasing concentration of ethanol. It was also found that the CD ellipticity at 222 nm, characteristic of the triple-helical structure, does not correlate with the thermal stability of collagen. Time-dependent experiments reveal that the collagen triple helix is kinetically stable in the presence of 0–40 % (v/v) ethanol at low temperature (5 °C) but highly unstable in the presence of ethanol at elevated temperature (~34 °C). These results indicate that when ethanol is used to process collagen-based biomaterials, such factors as temperature and duration should be done taking into account, to prevent extensive damage to the triple-helical structure of collagen .     

Crystalline structure of the gas vesicle wall from Anabaena flos-aquae.

The gas vesicles isolated from Anabaena flos-aquae have been studied by X-ray diffraction. Electron microscopy has previously shown that the gas vesicles are elongated shapes, with a thin wall having regular striations (ribs) at right-angles to the long axis. The X-ray diffraction pattern from a specimen of oriented, intact vesicles includes a number of sharp reflections which are attributed to regular structure in the plane of the wall. After correcting for the imperfect alignment of the long axes of the vesicles, the in-plane reflections are all seen to lie on a few, regularly spaced lines parallel to the long axis. This result shows for the first time that there are subunits regularly spaced along each rib, one subunit every 11 Å. The spacing of the in-plane reflections along each line is consistent with a rib periodicity of 46 Å. The 11 Å repeat, together with the 46 Å repeating distance from rib to rib and the average wall thickness of about 20 Å, define a volume for the subunit. Assuming a reasonable value for the density of the protein making up the wall, the molecular weight of the subunit indicated is about 8000 g/mol.The X-ray data also indicate that a large part of the protein is in the β-sheet conformation. In this structure there are parallel, or anti-parallel, polypeptide chains which are hydrogen-bonded to one another in a regular way to form a thin sheet. Assuming the wall contains β-sheet in two layers, one on top of the other and with the chains in each layer tilted at 35 ° to the long axis of the vesicle, we can explain a number of the X-ray observations: (1) oriented arcs with a Bragg spacing of 4.7 Å, which is the distance between the axes of neighbouring chains in each layer; (2) diffraction oriented in the direction of the chains at a spacing of 6 to 7 Å, which is the repeating distance of the dipeptide unit along the chain; (3) the 11 Å repeat, which is the repeating distance of pairs of chains along each rib; and (4) a broad band of diffraction at right-angles to the plane of the wall and centred at a spacing of 10 Å, which is a reasonable value for the distance between the mid-planes of the two sheets. Moreover, we can also find the remaining lattice parameter, the angle relating the centres of the subunits in neighbouring ribs. Thus the shortest line joining the centres makes an angle of 86 ° with the direction of the ribs.     

The variability in type I collagen helical pitch is reflected in the D periodic fibrillar structure

The variability in amino acid axial rise per residue of the collagen helix is a potentially important parameter that is missing in many structural models of fibrillar collagen to date. The significance of this variability has been supported by evidence from collagen axial structures determined by electron microscopy and X-ray diffraction, as well as studies of the local sequence-dependent conformation of the collagen helix. Here, sequence-dependent variation of the axial rise per residue was used to improve the fit between simulated diffraction patterns derived from model structures of the axially projected microfibrillar structure and the observed X-ray diffraction pattern from hydrated rat tail tendon. Structural models were adjusted using a genetic algorithm that allowed a wide range of structures to be tested efficiently. The results show that variation of the axial rise per residue could reduce the difference metric between model and observed data by up to 50%, indicating that such a variable is a necessary part of fibril model structure building. The variation in amino acid translation was also found to be influenced by the number of proline and hydroxyproline residues in the triple helix structure.     

On the structure of agglutinated sheep red blood cell membranes

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160–186 Å. It is concluded that two asymmetric membranes are contained inthe elementary period. Lipid phases with preferentialyl oriented hydrocarbon chains are part of the membrane structure. The stacking of membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 Å^?1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.     

胰蛋白酶消化法测定牛胶原蛋白三股螺旋结构的含量

本研究建立了一种测定胶原蛋白的三股螺旋结构含量的方法。该方法通过使用柱前衍生高效液相色谱(HPLC)法表征经胰蛋白酶酶解后胶原蛋白羟脯氨酸(Hyp)质量浓度的变化,进而对胶原蛋白的三股螺旋结构进行定量。探讨了不同的酶解时间(0~48h)、酶与底物的比例(1∶100、1∶50和1∶20)和温度(20、25、30、37℃)对明胶降解率的影响。获得了酶解的最佳条件——当胰蛋白酶与底物的比例为1∶50时,25℃酶解3h。使用该方法对明胶胶原蛋白混合液检测,结果表明,该方法能灵敏(RSD<10%)的测定胶原蛋白三股螺旋结构的含量。该方法不仅可用于生物组织研究领域,也可用于胶原蛋白食品、保健品和组织工程产品质量的评价。     

Filamentous Bacterial viruses

The filamentous bacterial virus is a simple and well-characterized model system for studying how genetic information is transformed into molecular machines. The viral DNA is a single-stranded circle coding for about 10 proteins. The major viral coat protein is largely α-helical, with about 46 amino acid residues. Several thousand identical copies of this protein in a helical array form a hollow cylindrical tube 1–2μ long, of outer diameter 60 Å and inner diameter 20 Å, with the twisted circular DNA extending down the core of the tube. Before assembly, the viral coat protein spans the cell membrane, and assembly involves extrusion of the coat from the membrane. X-ray fibre diffraction patterns of the Pf 1 species of virus at 4°C, oriented in a strong magnetic field, give three-dimensional data to 4 Å resolution. An electron density map calculated from native virus and a single iodine derivative, using the maximum entropy technique, shows a helix pitch of 5.9 Å. This may indicate a stretched A-helix, or it may indicate a partially 3₁₀ helix conformation, resulting from the fact that the coat protein is an integral membrane protein before assembly, and is still in the hydrophobic environment of other coat proteins after assembly.     

A 12 Å resolution X-ray diffraction study of the profile structure of isolated bovine retinal rod outer segment disk membranes

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 Å resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 Å. The diffraction patterns changed dramatically at 150 Å; consequently, the low (less than 150 Å) and high (greater than 150 Å) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.     

Gel formation from solutions of single-chain gelatin

The changes in conformation undergone by α-gelatin molecules on quenching aqueous solutions to below the temperature at which they can gel have been monitored by nuclear magnetic resonance and dielectric relaxation techniques. The relative rates of these conformational transitions are compared with changes in rheological properties. The nmr spectral intensity changes for 0.2 and 0.5% w/v α-gelatin solutions correspond to a unimolecular process with k ～ 10^?2 min^?1 at 15°C; this process occurs independently of whether or not the solution is concentrated enough to form a gel. The process involves a slow intramolecular nucleation step, followed by a rapid conformational change of the whole molecule from random coil to a rigid stage. Comparison with other data suggests that the transition gives rise to a triple collagen-like helix. In dilute solution (but above the critical concentration for gel formation, e.g., 0.5% w/v), the gelatin process follows the formation of the rigid molecular species. It probably involves the formation of junction zones consisting of three polypeptide chains in a collagen-like triple-helical conformation. These junctions may form, at low concentrations, from a reorganization of previously formed, intramolecular, triple helices. Solutions below a concentration of about 0.4% w/v α-gelatin cannot gel by this mechanism, and only form viscous liquids.     

Structural properties of a collagenous heterotrimer that mimics the collagenase cleavage site of collagen type I

Collagens contain sequence- and conformation-dependent epitopes responsible for their digestion by collagenases at specific loci. A synthetic heterotrimer construct containing the collagenase cleavage site of collagen type I was found to mimic perfectly native collagen in terms of selectivity and mode of enzymatic degradation. The NMR conformational analysis of this molecule clearly revealed the presence of two structural domains, i.e. a triple helix spanning the Gly-Pro-Hyp repeats and a less ordered portion corresponding to the collagenase cleavage site where the three chains are aligned in extended conformation with loose interchain contacts. These structural properties allow for additional insights into the very particular mechanism of collagen digestion by collagenases.