Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.     

Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree(Ginkgo biloba L.)

We studied in detail the mean micro fibril angle and the width of cellulose crystals from the pith to the bark of a 15-year-old Maidenhair tree(Ginkgo biloba L.). The orientation of cellulose micro fibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using Xray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean micro fibril angle,varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1–3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2 L layer con firmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The micro fibril angle remained large over the 14 annual rings. The entire stem disc,with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.     

Lamellar organization of pigments in chlorosomes, the light harvesting complexes of green photosynthetic bacteria

Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature. In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates. Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints. We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy. Cryoelectron microscopy images revealed dense striations approximately 20 A apart. X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing. No evidence for the rod models was obtained. The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays. The diffraction data further indicate that arrays are built from BChl dimers. The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids. The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.     

Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation

Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.     

Amorphous characteristics of an ultrathin cellulose film

Swelling behavior and rearrangements of an amorphous ultrathin cellulose film (20 nm thickness) exposed to water and subsequently dried were investigated with grazing incidence X-ray diffraction, neutron reflectivity, atomic force microscopy, and surface energy calculations obtained from contact angle measurements. The film swelled excessively in water, doubling its thickness, but shrunk back to the original thickness upon water removal. Crystallinity (or amorphousness) and morphology remained relatively unchanged after the wetting/drying cycle, but surface free energy increased considerably (ca. 15%) due to an increase in its polar component, that is, the hydrophilicity of the film, indicating that rearrangements occurred during the film's exposure to water. Furthermore, stability of the films in aqueous NaOH solution was investigated with quartz crystal microbalance with dissipation monitoring. The films were stable at 0.0001 M NaOH but already 0.001 M NaOH partially dissolved the film. The surprising susceptibility to dissolve in dilute NaOH was hypothetically attributed to the lack of hierarchical morphology in the amorphous film.     

Interactions of hydrophobin proteins in solution studied by small-angle X-ray scattering

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5°C-60°C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.     

Studies of crystallinity of Scots pine and Norway spruce cellulose

The variation in the mass fraction of crystalline cellulose (crystallinity of wood), the intrinsic crystallinity of cellulose, and the thickness of cellulose crystallites in early wood of Norway spruce [Picea abies (L.) Karst.], and Scots pine (Pinus sylvestris L.) grown in Finland were studied using wide angle X-ray scattering and nuclear magnetic resonance spectroscopy. The mass fraction of crystalline cellulose in wood increased slightly with the distance from the pith and was about 30±4% in mature wood of both species. The crystallinity of cellulose and the thickness of cellulose crystallites were almost constant for both species. The crystallinity of cellulose was 52±3% for both species and the average thickness of the cellulose crystallites was 32±1 Å and 31±1 Å for Norway spruce and Scots pine, respectively. The mass fraction of cellulose in wood, calculated from the crystallinity values, increased with the distance from the pith for both species.     

X-ray microdiffraction reveals the orientation of cellulose microfibrils and the size of cellulose crystallites in single Norway spruce tracheids

The microfibril angle (MFA) distribution and the size of cellulose crystallites in isolated double cell walls of Norway spruce (Picea abies [L.] Karst.) tracheids were determined by synchrotron X-ray microdiffraction using the reflections 200 and 004. Samples were 25 μm thick longitudinal sections of earlywood from annual rings 6–18 of several stems. The asymmetric MFA distributions extended from ?20° to 90°. The mean MFA of tangential cell walls decreased from an average of 24° into 19° from the pith to the bark. The mode of the MFA distribution was about 10° smaller than the mean MFA. The standard deviation of the MFA distribution varied between 18° and 25°. The mean MFA and the mode of the MFA distribution were larger in radial than in tangential cell walls. MFA distributions of mature wood samples exhibited a separate small peak at around 90°. The average width and length of cellulose crystallites varied between 28.9–30.9 Å and 192–284 Å, respectively. Both increased slightly as a function of annual ring number from the pith up to the 15th annual ring. An irrigation–fertilisation treatment of some of the stems resulted in longer cellulose crystallites compared to the untreated stems.     

Characterization of apo and partially saturated states of calerythrin, an EF-hand protein from S. erythraea: a molten globule when deprived of Ca(2+)

Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca(2+) and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca(2+)-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca(2+)-saturated states.     

Negative Poisson ratio of crystalline cellulose in kraft cooked Norway spruce

The tensile properties of kraft cooked Norway spruce were studied by tensile testing with in situ X-ray diffraction (XRD). Samples were of earlywood, cooked for varying times. The total lignin content of the samples was between 21.7% and 9.3%. Tensile tests with XRD were performed on wet samples, without XRD on dry samples. The tensile strength, the modulus of elasticity (MOE), and the elongation at fracture/yield were determined. X-ray diffraction was used to determine the microfibril angle (MFA) and the deformation of crystalline cellulose by monitoring the reflections 200 and 004. The (X-ray) Poisson ratio of crystalline cellulose was calculated, both before and after the yield point. The tensile strength and the MOE of the wet samples were significantly lower than in the dry samples. The tensile properties of dry samples were similar to dry earlywood samples of untreated Norway spruce. The MFA only showed notable changes due to strain when it was initially large, when a diminishing effect was observed. The Poisson ratio of crystalline cellulose was negative. The average values ranged between -0.26 and -1.17 before the yield point and between -0.86 and -1.05 after the yield point.