Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

Effects of pH and ionic strength on the structure of collagen fibrils

The roles of pH and ionic strength on the structure and stability of collagen fibrils have been investigated by means of x-ray and neutron diffraction techniques. High-angle x-ray diffraction shows that a salt concentration of 0.5M KCl is sufficient to reduce the osmotic swelling and related disordering in the pH range 1–3. The relative intensities of the low-angle meridional x-ray and neutron diffraction Bragg reflections vary with pH. Difference Fourier syntheses between pH 7 and 1.6 data indicate, for both x-ray and neutron diffraction, a reduced scattering contribution from the telopeptides at low pH. Lyotropic relaxation is a crucial step in the appearance at low pH of a doubling of the 668-Å axial periodicity (D) of collagen fibrils. These results suggest that electrostatic interactions are essential for the structural stability of the telopeptide regions and of the 1D and 3D intermolecular staggers between collagen molecules.     

Electron diffraction and high-resolution imaging on highly-crystalline β-chitin microfibril

The ultrastructure of β-chitin microfibrils from a centric diatom, Thalassiosira, and a tubeworm, Lamellibrachia, was studied using electron diffraction and high-resolution electron microscopy. Electron microdiffraction diagrams corresponding to each projection of the β-chitin crystals were obtained, and all the data support the structure model of anhydrous β-chitin crystals proposed by X-ray diffraction experiments. From high-resolution electron microscopy on ultrathin sections, the cross-sectional shapes of the microfibrils from Thalassiosira and Lamellibrachia were observed as a rectangular and parallelogram, respectively. The lattice fringes corresponding to the (0 1 0) plane of anhydrous β-chitin crystals were clearly observed in both cross-sections. Based on these observations, we have constructed a molecular packing model for β-chitin microfibrils.     

Periodic disorder along ramie cellulose microfibrils

Small angle neutron scattering studies have been carried out on cellulose fibers from ramie and Populus maximowicii (cotton wood). Labile hydrogen atoms were replaced by deuterium atoms, in water-accessible disordered regions of the fibers, to increase the neutron scattering contrast between the disordered and crystalline regions. A meridional Bragg reflection, corresponding to a longitudinal periodicity of 150 nm, was observed when scattering collected from hydrogenated and deuterated dry ramie fibers was subtracted. No Bragg reflection was observed with the cotton wood fibers, probably because of lower orientation of the microfibrils in the cell wall. The ramie fibers were then subjected to electron microscopy, acid hydrolysis, gel permeation chromatography, and viscosity studies. The leveling off degree of polymerization (LODP) of the hydrolyzed samples matched exactly the periodicity observed in the diffraction studies. The weight loss related to the LODP was only about 1.5%, and thus, the microfibrils can be considered to have 4-5 disordered residues every 300 residues.     

Synthèse et structure de la poly-O-carbobenzoxy L- tyrosine et de copolymères de O-carbobenzoxy L-tyrosine et de glutamate de benzyle ou d'aspartate de benzyle

The optical rotatory dispersion of copolymers of O-carbobenzoxy-L -tyrosine and benzyl L (or D )-glutamate as well as benzyl L -aspartate, dissolved in nonpolar solvent, has been studied. Moffitt's equation permits the determination of b₀ coefficients whose variation, with varying composition in amino acid residues, suggests that the molecules of poly-O-carbobenzoxy-L -tyrosine have a helical structure similar to that of poly-(benzyl L -glutamate). Results obtained from infrared spectroscopy and x-ray diffraction show that the copolymers possess a helical conformation in the solid state, even when they are very rich in carbobenzoxy-L -tyrosine residues. The value of the b₀, coefficient for poly-O-carbobenzoxy-L -tyrosine may be explained by a regular stacking of the chromophore groups around the helical backbone. The ordering of the molecules of this polymer in a purely helical structure seems favored by the insertion of a small number of foreign residues in the polypeptide chain.     

Raman spectra of D and L amino acid copolymers. Poly-DL-alanine, poly-DL-leucine, and poly-DL-lysine.

The Raman spectrum of poly-DL -alanine (PDLA) in the solid state is interpreted in terms of the disordered chain conformation, in analogy with the spectrum of mechanically deformed poly-L -alanine. The polymer is largely disordered with only a small α-helical content in the solid state. When PDLA is dissolved in water, the spectra suggest that short α-helical segments are formed upon dissolution. These helical regions might be stabilized by hydrophobic bonds between side-chain methyl groups. Addition of methanol to the aqueous PDLA solutions results in a Raman spectrum resembling that of solid PDLA. This result suggests that the methanol disrupts the helical regions by breaking the hydrophobic bonds. The Raman spectra of poly-DL -leucine (PDLL) and poly-L -leucine (PLL) are compared and only slight differences are observed in the amide I and III regions, indicating that PDLL does not have an appreciable disordered chain content. Significant differences are observed in the skeletal regions. The 931-cm^?1 lines in the PLL and PDLL spectra are assigned to residues in α-helical segments of the preferred screw sense, i.e., L -residues in right-handed segments and D -residues in left-handed segments (in PDLL). On the other hand, the 890-cm^?1 line in the spectrum of PDLL is assigned to residues not in the preferred helical sence, i.e., L -residues in left-handed segments and D -residues in right-handed ones. The Raman spectra of poly-DL -lysine and poly-L -lysine in salt-free water at pH 7.0 are compared. The Raman spectra of the two polymers are very similar. However, this does not negate the hypothesis of local order in poly-L -lysine because the distribution of the residues in poly-DL -lysine probably tends towards blocks, and the individual blocks may take up the 3₁ helix.     

Mechanical behavior of cellulose microfibrils in tension wood, in relation with maturation stress generation

A change in cellulose lattice spacing can be detected during the release of wood maturation stress by synchrotron x-ray diffraction experiment. The lattice strain was found to be the same order of magnitude as the macroscopic strain. The fiber repeat distance, 1.033 nm evaluated for tension wood after the release of maturation stress was equal to the conventional wood values, whereas the value before stress release was larger, corresponding to a fiber repeat of 1.035 nm, nearly equal to that of cotton and ramie. Interestingly, the fiber repeat varied from 1.033 nm for wood to 1.040 nm for algal cellulose, with an increasing order of lateral size of cellulose microfibrils so far reported. These lines of experiments demonstrate that, before the stress release, the cellulose was in a state of tension, which is, to our knowledge, the first experimental evidence supporting the assumption that tension is induced in cellulose microfibrils.     

Refined atomic model of the four-layer aggregate of the tobacco mosaic virus coat protein at 2.4-A resolution.

Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.     

Lattice images from ultrathin sections of cellulose microfibrils in the cell wall of Valonia macrophysa Kütz.

The crystalline ultrastructure and orientation of cellulose microfibrils in the cell wall of Valonia macrophysa were investigated by means of high-resolution electron microscopy of ultrathin (approx. 28 nm) sections. With careful selection of imaging conditions, ultrastructural aspects of the cell wall that had remained unresolved in previous studies were worked out by direct imaging of crystal lattice of cellulose microfibrils. It was confirmed that each microfibril is a single crystal having a lateral dimension of 20·20 nm², because lattice images of 0.39 nm resolution were clearly recorded with no major disruption in the whole area of the cross section of the microfibril. There was no evidence for the existence of 3.5-nm elementary fibrils which have been considered to be basic crystallographic and morphological units of cellulose in general. It was also confirmed that the axial directions (crystallographic fiber direction) of adjacent microfibrils in each single lamella of the cell wall are opposite to each other.     

Cellulose microfibril assembly inErythrocladia subintegra Rosenv.: An ideal system for understanding the relationship between synthesizing complexes (TCs) and microfibril crystallization

Summary The marine red algaErythrocladia subintegra synthesizes cellulose microfibrils as determined by CBH I-gold labelling, X-ray and electron diffraction analyses. The cellulose microfibrils are quite thin, ribbon-like structures, 1–1.5 nm in thickness (constant), and 10–33 nm in width (variable). Several laterally associated minicrystal components contribute to the variation in microfibrillar width. Electron diffraction analysis suggested a uniplanar orientation of the microfibrils with their (101) lattice planes parallel to the plasma membrane surface of the cell. The linear particle arrays bound in the plasma membrane and associated with microfibril impressions recently demonstrated inErythrocladia have been shown in this study to be the cellulose-synthesizing terminal complexes (TCs). The TCs appear to be organized by a repetition of transverse rows consisting of four TC subunits, rather than by four rows of longitudinallyarranged TC subunits. The number of transverse rows varied between 8–26, corresponding with variation in the length of the TCs and the width of the microfibrils. The spacings between the neighboring transverse rows are almost constant being 10.5–11.5 nm. Based on the knowledge thatAcetobacter, Vaucheria, andErythrocladia synthesize similar thin, ribbon-like cellulose microfibrils, the structural characteristics common to the organization of distinctive TCs occurring in these three organisms has been discussed, so that the mode of cellulose microfibril assembly patterns may be deciphered.     

Electron diffraction from the primary wall of cotton fibers

Summary Electron diffraction patterns have been obtained from selected areas of disencrusted microfibrils isolated from the primary cell wall of cotton fibers. The resultant fiber diagram has the same meridional repeat distance as a corresponding pattern of secondary wall microfibrils but differs markedly in the equatorial reflections. The primary wall diagram displays only two strong equatorial reflections centered at 0.570 nm and 0.416 nm. The similarity of these spacings with those of cellulose IV suggests that the crystalline structure of the primary wall cellulose is similar to that of cellulose IV_I and is best explained in term of native cellulose I crystals having good longitudinal coherence (i.e., coherence along the length of the microfibrils) but with poor lateral organization of the network of inter chain hydrogen bonds. Similar results were also obtained for other primary wall specimens.     

Calcium Determines the Supramolecular Organization of Fibrillin-rich Microfibrils

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of ~56 nm. A Ca²⁺-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca²⁺. In the presence of Ca²⁺, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca²⁺ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca²⁺ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca²⁺-containing, staggered microfibrillar arrays were used to interpret the effects of Ca²⁺. These observations highlight the importance of Ca²⁺ to microfibrils and microfibrillar arrays in vivo.     

Conformational aspects of polypeptides. XXV. Solvent and temperature effects on the conformations of copolymers of benzyl and methyl L-aspartate with nitrobenzyl L-aspartate

A series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate and with β-mcthyl L -aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.     

Role of two consecutive α,β-dehydrophenylalanines in peptide structure: Crystal and molecular structure of Boc-Leu-ΔPhe-ΔPhe-Ala-Phe-NHMe

An N^α-protected model pentapeptide containing two consecutive ΔPhe residues, Boc-Leu-ΔPhe-ΔPhe-Ala-Phe-NHMe, has been synthesized by solution methods and fully characterized. ¹H-nmr studies provided evidence for the occurrence of a significant population of a conformer having three consecutive, intramolecularly H-bonded β-bends in solution. The solid state structure has been determined by x-ray diffraction methods. The crystals grown from aqueous methanol are orthorhombic, space group P2₁2₁2₁, a = 11.503(2), b = 16.554(2), c = 22.107(3) Å, V = 4209(1) Å,³ and Z = 4. The x-ray data were collected on a CAD4 diffractometer using CuK_a radiation (λ = 1.5418 Å). The structure was determined using direct methods and refined by full-matrix least-squares procedure. The R factor is 5.3%. The molecule is characterized by a right handed 3₁₀-helical conformation (〈ϕ〉 = −68.2°, 〈ψ〉 = −26.3°), which is made up of two consecutive type III β-bends and one type I β-bend. In the solid state the helical molecules are aligned head-to-tail, thus forming long rod like structures. A comparison with other peptide structures containing consecutive ΔPhe residues is also provided. The present study confirms that the -ΔPhe-ΔPhe-sequence can be accommodated in helical structures. © 1997 John Wiley & Sons, Inc. Biopoly 42: 373–382, 1997     

The intermolecular space of reconstituted collagen fibrils

The extent, geometry and heterogeneity of the intermolecular space of hydrated, purified and reconstituted steer skin collagen fibrils has been characterized. The extent of the space has been assessed experimentally by an X-ray diffraction method and a new physical chemical technique, and found to be 1.14 ml per gram collagen. A theoretical model relating the intermolecular space to X-ray diffraction parameters has been presented, and this suggests that the geometry of the intermolecular space arises from a near-hexagonal packing of the collagen molecules. On the basis of an assumed microfibrillar packing model and a geometric construction of the shape of a collagen molecule, the distribution of the space within reconstituted collagen fibrils has been characterized as follows: 0.13 ml of the intermolecular space/g collagen can be attributed to the helical groove of the collagen molecules per se and 1.01 ml/g is interstitial; 0.66 ml/g is present in the form of “pores” (hexagonally-closed packed spaces), whereas 0.48 ml/g is present in the form of “holes” (hexagonal volume defects); 0.73 ml/g of the intermolecular space is associated with a region of the collagen fibrils where holes are localized and 0.41 ml/g is attributable to the regions of the fibril in which pores only are present.     

The Cellulose System in the Cell Wall ofMicrasterias

The cellulose system of the cell wall ofMicrasterias denticulataandMicrasterias rotatawas analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose ofMicrasteriasis very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having ad-spacing of 0.60 nm [(110) in the Iβ cellulose unit cell defined by Sugiyamaet al.,1991,Macromolecules24, 4168–4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only inSpirogyra,another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose ofMicrasteriasmay be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes.     

Biophysical studies on fragments of the α-factor receptor protein

The receptor for the α-factor mating pheromone of the yeast Saccharomyces cerevisiae consists of 431 amino acid residues and is a member of a family of membrane proteins predicted to have seven transmembrane helices. Fragments of the receptor corresponding to two of the transmembrane helices [residues 246–269 (M6) and 273–302 (M7)], two of the interhelical loops [residues 107–125 (E2) and 191–206 (E3)], and to a portion of the carboxyl terminus [residues 350–372 (CT)] were synthesized using solid-phase methodologies and purified to near homogeneity. CD was used to characterize the secondary structure of these peptides in trifluoroethanol (TFE), in TFE/water mixtures, in sodium dodecyl sulfate (SDS), and in the presence of dimyristoyl phosphatidylcholine (DMPC) liposomes. In TFE, M6 and M7 exhibited CD spectra consistent with highly helical peptides, whereas CT was partially helical. In contrast, E2 and E3 were either disordered or aggregated in this solvent. M6 did not partition well into DMPC vesicles whereas M7 remained helical. Both M6 and M7 assumed helical conformations in 25 mM SDS. The loop neptides and the carboxyl terminus peptide were either in a β-structure or disordered in the presence of lipid. These findings represent the first biophysical evidence for conformations assumed by specific segments of the STE2 receptor protein. © 1994 John Wiley & Sons, Inc.     

The cyanide hydratase from <Emphasis Type="Italic">Neurospora crassa</Emphasis> forms a helix which has a dimeric repeat

The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution (3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D₁ S_5.4 symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic model into the experimentally determined negative stain envelope suggests the location of charged residues which may form salt bridges and stabilize the helix.     

Length-dependent prediction of protein intrinsic disorder

Background  

Due to the functional importance of intrinsically disordered proteins or protein regions, prediction of intrinsic protein disorder from amino acid sequence has become an area of active research as witnessed in the 6th experiment on Critical Assessment of Techniques for Protein Structure Prediction (CASP6). Since the initial work by Romero et al. (Identifying disordered regions in proteins from amino acid sequences, IEEE Int. Conf. Neural Netw., 1997), our group has developed several predictors optimized for long disordered regions (>30 residues) with prediction accuracy exceeding 85%. However, these predictors are less successful on short disordered regions (≤30 residues). A probable cause is a length-dependent amino acid compositions and sequence properties of disordered regions.     

Interaction of an Amphiphilic Peptide with a Phospholipid Bilayer Surface by Molecular Dynamics Simulation Study

Abstract

Corticotropin-releasing factor (CRF) is the principal neuroregulator of adrenocorticotropic hormone (ACTH) secretion. Previous experiments have demonstrated that CRF binds avidly to the surface of single egg phosphatidylcholine vesicles and its amphiphilic secondary structure might play an important role in the function. In this study, the interaction of the residues 13–41 in human CRF with the surface of a DOPC bilayer was investigated by molecular dynamics (MD) simulation in order to understand the role of the membrane surface in the formation of the amphiphilic α helix as well as to determine the effects of the peptide on the lipid bilayer. The model used included 60 DOPC molecules, 1 helical peptide (CRF_13–41) on the bilayer surface, and explicit waters of solvation in the lipid polar head group regions, together with constant-volume periodic boundary conditions in three dimensions. The MD simulation was carried out for 510 ps. In addition, CRF_13–41, initially in a helical form, was simulated in vacuo as a control. The results indicate that while it was completely unstable in vacuo, the peptide helical form was generally maintained on the bilayer surface, but with distortions near the terminal ends. The peptide was confined to the bilayer headgroup/water region, similar to that reported from neutron diffraction measurement of tripeptides bound to the phosphatidylcholine bilayer surface (Ref 1). The amphiphilicity of the peptide matched that of the bilayer headgroup environment, with the hydrophilic side oriented toward water and the hydrophobic side making contact with the bilayer hydrocarbon core. These results support the hypothesis that the amphiphilic environment of a membrane surface is important in the induction of peptide amphiphilic α-helical secondary structure. Two major effects of the peptide on the lipids were found: the first CH₂ segment in the lipid chains was significantly disordered and the lipid headgroup distribution was broadened towards the water region.