Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice.

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.

Preferred side-chain conformation of arginine residues in a triple-helical structure.

The crystal structure of the triple-helical peptide (Pro-Hyp-Gly)3 -Pro-Arg-Gly-(Pro-Hyp-Gly)4 (POG3-PRG-POG4) was determined at 1.45 Å resolution. POG3-PRG-POG4 was designed to permit investigation of the side-chain conformation of the Arg residues in a triple-helical structure. Because of the alternative structure of one of three Arg residues, four side-chain conformations were observed in an asymmetric unit. Among them, three adopt a ttg(-) t conformation and the other adopts a tg(-) g(-) t conformation. A statistical analysis of 80 Arg residues in various triple-helical peptides showed that, unlike those in globular proteins, they preferentially adopt a tt conformation for χ1 and χ2 , as observed in POG3-PRG-POG4. This conformation permits van der Waals contacts between the side-chain atoms of Arg and the main-chain atoms of the adjacent strand in the same molecule. Unlike many other host-guest peptides, in which there is a significant difference between the helical twists in the guest and the host peptides, POG3-PRG-POG4 shows a marked difference between the helical twists in the N-terminal peptide and those in the C-terminal peptide, separated near the Arg residue. This suggested that the unique side-chain conformation of the Arg residue affects not only the conformation of the guest peptide, but also the conformation of the peptide away from the Arg residue.

Crystal structure of the collagen model peptide (Pro-Pro-Gly)4-Hyp-Asp-Gly-(Pro-Pro-Gly)4 at 1.0 Å resolution.

The single-crystal structure of the collagen-like peptide (Pro-Pro-Gly)4 -Hyp-Asp-Gly-(Pro-Pro-Gly)4, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple-helical structure and that its conformation is very similar to that of (Gly-Pro-Hyp)9, which has the typical repeating sequence in collagen. High-resolution studies on other collagen-like peptides have shown that imino acid-rich sequences preferentially adopt a 7/2 triple-helical structure (θ = 51.4°), whereas imino acid-lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly-Hyp-Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro-Pro-Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2-helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N-terminus of an adjacent molecule, causing axial displacement, reminiscent of the D-staggered structure in fibrous collagens.

Two crystal modifications of (Pro-Pro-Gly)4-Hyp-Hyp-Gly-(Pro-Pro-Gly)4 reveal the puckering preference of Hyp(X) in the Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs in collagen helices.

Two crystal modifications of a collagen model peptide, (Pro-Pro-Gly)(4)-Hyp-Hyp-Gly-(Pro-Pro-Gly)(4) [where Hyp is (4R,2S)-L-hydroxyproline], showed very similar unit-cell parameters and belonged to the same space group P2(1). Both crystals exhibited pseudo-merohedral twinning. The main difference was in their molecular-packing arrangements. One modification showed pseudo-hexagonal packing, while the other showed pseudo-tetragonal packing. Despite their different packing arrangements, no significant differences were observed in the hydration states of these modifications. The peptide in the pseudo-tetragonal crystal showed a cyclic fluctuation of helical twists with a period of 20 A, while that in the pseudo-hexagonal crystal did not. In these modifications, the puckering conformations of four of the 12 Hyp residues at the X position of the Hyp(X)-Hyp(Y)-Gly sequence were in the opposite conformations to the previous hypothesis that Hyp(X) residues involved in Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs prefer up-puckering and down-puckering conformations, respectively. Detailed investigation of the molecular interactions between Hyp(X) and adjacent molecules revealed that these opposite conformations appeared because the puckering conformation, which follows the hypothesis, is subject to steric hindrance from the adjacent molecule.

Complexation of syndiotactic polystyrene with 12-crown-4.

Solid-state complexation of syndiotactic polystyrene (sPS) with a crown ether compound, 1,4,7,10-tetraoxa-cyclododecane (12-crown-4), took place when a film of sPS/chloroform clathrate was subjected to a guest exchange procedure assisted with a plasticizing agent. The new guest 12-crown-4 molecules were incorporated into the crystalline region of the sPS film, without causing a large conformational change of host sPS helices. X-ray diffraction and thermogravimetric investigations showed that sPS/12-crown-4 complex had a clathrate complex structure which contained four 12-crown-4 molecules per unit cell. IR and Raman data suggested that 12-crown-4 took a C(i) -type conformation in the sPS complex phase.

Dynamics and hydration behavior of a short collagen model polypeptide, (L-prolyl-L-prolylglycyl)5, in aqueous media.

A short collagen model polypeptide, (l-prolyl-l-prolylglycyl)(5) (PPG5), behaves as a fully dissociated flexible zwitterionic polymer chain in pure water. Its first and third normal modes of chain conformational fluctuation were detected as distinct dielectric relaxation modes. Addition of acetic acid at 30 mM to an aqueous solution of PPG5 effectively suppressed the overall relaxation strength by protonation of the carboxy termini of the polypeptide. Furthermore, the hydration number per PPG5 molecule was determined to be 130 by dielectric relaxation (DR) spectroscopy over a frequency range from 1 MHz to 20 GHz; this value was not affected by the addition of acetic acid.

Revisiting the molecular structure of collagen.

The triple helix is a specialized protein motif found in all collagens. Although X-ray diffraction studies of collagen began in the 1920s, the very small amount of data available from fiber diffraction of native collagen caused the determination of its molecular conformation to take a very long time. In the early 1950s, two plausible fiber periods of about 20 and 30 A were proposed, together with corresponding single-strand models having 7/2- and 10/3-helical symmetry, respectively. The first framework of the triple helix was proposed by Ramachandran and Kartha in 1955. In the same year, Rich and Crick proposed another structure with the same framework that avoided some of the steric problems of the first model. Their framework, which involved a triple-helical structure with a fiber period of 28.6 A and 10/3-helical symmetry, was exactly the same as one of two single-strand models for collagen proposed at that time, except for the number of strands. At that time, however, nobody considered the triple-strand model with the other framework, with a fiber period of 20 A and 7/2-helical symmetry, until Okuyama et al. detected this structure in the single crystal of (Pro-Pro-Gly)(10) in 1972. Although they proposed this structure as a new structural model for collagen in 1977, it has not been acknowledged as such, but instead has been regarded only as a model for a collagen-like peptide. In 2006, it was shown that both 7/2- and 10/3-helical models could explain X-ray diffraction data from native collagen quantitatively. Furthermore, during the past decade, many single crystals of collagen-model peptides have been analyzed at high resolution. The helical symmetries observed in these model peptides are very close to the ideal 7/2-helical symmetry, whereas no supporting data were found for the 10/3-helical model. This evidence strongly suggests that an average molecular structure of native collagen is the 7/2-helical model rather than the prevailing Rich and Crick (10/3-helical) model. Knowing the correct molecular structure, the driving force for the formation of a quarter-staggered structure in collagen fibrils will be elucidated in the near future by analysis incorporating the molecular structure of collagen and its amino acid sequence.

Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon.

In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix. Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules. A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix. The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface.

Repetitive interactions observed in the crystal structure of a collagen-model peptide, [(Pro-Pro-Gly)9]3.

The crystal structure of a collagen-model peptide [(Pro-Pro-Gly)(9)](3) has been determined at 1.33 A resolution. Diffraction data were collected at 100 K using synchrotron radiation, which led to the first structural study of [(Pro-Pro-Gly)(n)](3) under cryogenic conditions. The crystals belong to the P2(1) space group with cell parameters of a = 25.95, b = 26.56, c = 80.14 Angstroms and beta = 90.0 degrees. The overall molecular conformation was consistent with the left-handed 7/2-helical model with an axial repeat of 20 A for native collagen. A total of 332 water molecules were found in an asymmetric unit. Proline residues in adjacent triple-helices exhibited three types of hydrophobic interactions. Furthermore, three types of hydrogen-bonding networks mediated by water molecules were observed between adjacent triple-helices. These hydrophobic interactions and hydrogen-bonding networks occurred at intervals of 20 Angstroms along the c-axis based on the previous sub-cell structures [(Pro-Pro-Gly)(n)](3) (n = 9, 10), which were also seen in the full-cell structure of [(Pro-Pro-Gly)(10)](3). Five proline residues at the Y position in the X-Y-Gly triplet were found in a down-puckering conformation, this being inconsistent with the recently proposed propensity-based hypothesis. These proline residues were forced to adopt opposing puckering because of the prevailing hydrophobic interaction between triple-helices compared with the Pro:Pro stacking interaction within a triple-helix.

WinLALS for a linked-atom least-square refinement program for helical polymers on WINDOWS PCs.

Fiber diffraction dada from polymers are sufficiently different in kind and quantity from single crystal data as to warrant analyses with a different emphasis: refinement of competing molecular models where torsion angles and bond angles are the explicit variables rather than atomic coordinates. The first linked-atom least-squares (LALS) refinement program had been devolved at Arnott's laboratories at King's College London [Arnott, S., Wonacott, A.J., 1966a. Polymer 7, 157] on mainframe and several revised versions were maintained at Purdue University [Smith, P.J.C., Arnott, S., 1978. Acta Crystallogr. Sect. A 34, 3; Chandrasekan, R. 2000. LALS Users Manual, Whistler Centre for Carbohydrate Researchm Purdue University, West Lafayette, IN] on workstation. Today the LALS users have to choose correctly any one program that they want to use, trigonometric or Bessel functions, from some versions. To develop a new WinLALS program based on the dimensioned version of the latest LALS2000 program [LALS Users Manual (2000)], we reviewed all the mathematical expressions and corrected the optimization of the non-bonded atomic contact terms. The WinLALS is coded with FORTRAN 90 and runs on MICROSOFT-WINDOWS PCs and the many amendments including changing input/output assignments, expanding array sizes, arranging that the update files have all output parameters of each cycle, and correcting several bugs are performed. This paper describes the mathematical expressions in detail employed in WinLALS and compares results of its applications obtained with those obtained earlier.

Plausible molecular and crystal structures of chitosan/HI type II salt.

Chitosan/HI type II salt prepared from crab tendon was investigated by X-ray fiber diffraction. Two polymer chains and 16 iodide ions (I(-)) crystallized in a tetragonal unit cell with lattice parameters of a = b = 10.68(3), c (fiber axis) = 40.77(13) A, and a space group P4(1). Chitosan forms a fourfold helix with a 40.77 A fiber period having a disaccharide as the helical asymmetric unit. One of the O-3... O-5 intramolecular hydrogen bonds at the glycosidic linkage is weakened by interacting with iodide ions, which seems to cause the polymer to take the 4/1-helical symmetry rather than the extended 2/1-helix. The plausible orientations of two O-6 atoms in the helical asymmetric unit were found to be gt and gg. Two chains are running through at the corner and the center of the unit cell along the c-axis. They are linked by hydrogen bonds between N-21 and O-61 atoms. Two out of four independent iodide ions are packed between the corner chains while the other two are packed between the corner and center chains when viewing through the ab-plane. The crystal structure of the salt is stabilized by hydrogen bonds between these iodide ions and N-21, N-22, O-32, O-61, O-62 of the polymer chains.

Molecular and crystal structures of chitosan/HI type I salt determined by X-ray fiber diffraction.

The three-dimensional structure of chitosan/HI type I salt was determined by the X-ray fiber diffraction technique and linked-atom least-squares refinement method. Two polymer chains and four iodide ions (I(-)) crystallized in a monoclinic unit cell with dimensions a = 9.46(2), b = 9.79(2)], c (fiber axis)=10.33(2)A, beta = 105.2(2) degrees and a space group P2(1). Chitosan chains adopted an extended twofold helical conformation that was stabilized by O-3...O-5 hydrogen bonds, and the O-6 atom adopted nearly gt orientation. Polymer chains zigzag along the b-axis and directly connect to each other by N-2...O-6 hydrogen bonds. Two columns of iodide ions were shown to pack at the bending points of the zigzag sheets, and their locations are closely related to those of water columns in the hydrated chitosan. The iodide ions stabilized the salt structure by forming hydrogen bonds with the N-2 and O-6 atoms of the polymer chains together with an electrostatic interaction between N-2 and the iodide ions.

Two different molecular conformations found in chitosan type II salts.

The type II structure of chitosan acidic salts prepared from crab tendon in solid state was studied using an X-ray fiber diffraction technique together with the linked-atom least-squares (LALS) technique. The cylindrical Patterson method was applied to confirm the molecular conformation of the chitosan. It was shown that there are two different helical conformations for type II salts. One is the relaxed twofold helix having a tetrasaccharide as an asymmetric unit as found in chitosan.HCl salt, which was previously reported as a conformation of chitosan.HCOOH salt. The other is the fourfold helix having a disaccharide as an asymmetric unit newly found in chitosan.HI salt.

Three D structures of chitosan.

Crystal structures of two polymorphs of chitosan, tendon (hydrated) and annealed (anhydrous) polymorphs, have been reported. In both crystals, chitosan molecule takes up similar conformation (Type I form) to each other, an extended two-fold helix stabilized by intramolecular O3-O5 hydrogen bond, which is also similar to the conformation of chitin or cellulose. Three chitosan conformations other than Type I form have been found in the crystals of chitosan-acid salts. In the salts with acetic and some other acids, called Type II salts, chitosan molecule takes up a relaxed two-fold helix composed of asymmetric unit of tetrasaccharide. This conformation seems to be unstable because no strong intramolecular hydrogen bond like Type I form. Type II crystal changes to the annealed polymorph of chitosan by a spontaneous water-removing action of the acid. Chitosan molecule in its hydrogen iodide salt prepared at low temperature takes a 4/1 helix with asymmetric unit of disaccharide. The fourth chitosan conformation was found to be a 5/3 helix in chitosan salts with medical organic acids having phenyl group such as salicylic or gentisic acids. Similar conformation of chitosan molecule in the aspirin (acetylsalicylic acid) salt was suggested by a solid-sate NMR measurement.

Formation of Giant Liposomes from Crystalline Complexes of Monoalkylammonium Surfactants and 4-Hydroxybiphenyl.

A parallel rather than a perpendicular alignment of aromatic compounds with respect to surfactant molecules (see schematic representation) is preferred for the formation of a liposome structure, because the perpendicular alignment would reduce the hydrophilicity of the aggregate. This is the result of studies on crystalline complexes of monoalkylammonium halides and various aromatic compounds.

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.

Stabilization of triple-helical structures of collagen peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence.

The single-crystal structures of three collagen-like host-guest peptides, (Pro-Pro-Gly)(4) -Hyp-Yaa-Gly-(Pro-Pro-Gly)(4) [Yaa = Thr, Val, Ser; Hyp = (4R)-4-hydroxyproline] were analyzed at atomic resolution. These peptides adopted a 7/2-helical structure similar to that of the (Pro-Pro-Gly)(9) peptide. The stability of these triple helices showed a similar tendency to that observed in Ac-(Gly-Hyp-Yaa)(10) -NH(2) (Yaa = Thr, Val, Ser) peptides. On the basis of their detailed structures, the differences in the triple-helical stabilities of the peptides containing a Hyp-Thr-Gly, Hyp-Val-Gly, or Hyp-Ser-Gly sequence were explained in terms of van der Waals interactions and dipole-dipole interaction between the Hyp residue in the X position and the Yaa residue in the Y position involved in the Hyp(X):Yaa(Y) stacking pair. This idea also explains the inability of Ac-(Gly-Hyp-alloThr)(10) -NH(2) and Ac-(Gly-Hyp-Ala)(10) -NH(2) peptides to form triple helices. In the Hyp(X):Thr(Y), Hyp(X):Val(Y), and Hyp(X):Ser(Y) stacking pairs, the proline ring of the Hyp residues adopts an up-puckering conformation, in agreement with the residual preference of Hyp, but in disagreement with the positional preference of X in the Gly-Xaa-Yaa sequence.

Structure, dynamics, and hydration of a collagen model polypeptide, (L-prolyl-L-prolylglycyl)10, in aqueous media: a chemical equilibrium analysis of triple helix-to-single coil transition.

The structure, dynamics, and hydration behavior of a collagen model polypeptide, (L-prolyl-L-prolylglycyl)(10) (PPG10), were investigated in pure water and dilute acetic acid over a wide temperature range using broadband dielectric relaxation (DR) techniques that spanned frequencies from 1 kHz to 20 GHz. All samples showed pronounced dielectric dispersion with two major relaxation processes around 3 MHz and 20 GHz. Because DR measurements sensitively probe dipoles and their dynamics, the structures and ionization states of the carboxy and amino termini of aqueous PPG10 were precisely determined from the relaxation times and strengths in the 3 MHz frequency range. In solution, PPG10 formed mixtures of monodisperse rods as triple helices with lengths and diameters of 8.6 and 1.5 nm, respectively, and monomeric random coils with radii of approximately 1.4 nm. Ionization of the C-terminus was suppressed by the addition of acetic acid in both states. The fraction of random coils (f(coil)) was found to be a function of temperature (T) and the concentration of PPG10 (c). At low temperatures, small f(coil) values were found, which increased with temperature to reach f(coil) = 1 at approximately 60 degrees C, irrespective of c. This phenomenon, well-known as a triple helix-to-single coil transition, is discussed on the basis of the chemical reaction, (PPG10)(3) <==> 3PPG10, with an equilibrium constant of K = 3(c/55.6)(2)f(coil)(3)(1 - f(coil))(-1). The standard enthalpy change evaluated from Arrhenius plots (ln K versus T(-1)) was found to change dramatically at the same transition temperature that was previously determined by using optical rotation experiments. The other major DR process, observed at approximately 20 GHz, was assigned to free and hydrated water molecules and used to determine the average hydration number (m) per PPG10. The m values for the triple helix and random coil state at 25 degrees C were evaluated to be m(th) = 60-70 and m(coil) = 250-270. The m(th) value was in reasonable agreement with the number of hydrated water molecules in crystals of (PPG10)(3) residing in the first and second hydration shells around the amino acid residues. This agreement suggests that the structure of the triple helix in crystals is very similar to that in aqueous solution, including the location of hydrated water molecules.

High-resolution structures of collagen-like peptides [(Pro-Pro-Gly)4-Xaa-Yaa-Gly-(Pro-Pro-Gly)4]: implications for triple-helix hydration and Hyp(X) puckering.

Structures of (Pro-Pro-Gly)4-Xaa-Yaa-Gly-(Pro-Pro-Gly)4 (ppg9-XYG) where (Xaa, Yaa)=(Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro-Pro-Gly)9 peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down-puckering conformation, as in ppg9-OPG, and ppg9-OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up-puckering conformation if there is no specific reason to adopt the down-puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9-XYG peptides is very large, likely due to the unique rod-like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures.