An X-ray diffraction study of poly-L-arginine hydrochloride

An x-ray study has been made of polyarginine hydrochloride to investigate whether, like polylysine hydrochloride, it can undergo conformational changes merely from variations in the degree of hydration. X-ray powder and fiber photographs of specimens containing up to about five molecules of water per arginine residue show features characteristic of α-helical structures including a 5.4-Å layer line and a meridional 1.5-Å reflection. Increasing the water content from ¹/₂ to 6¹/₂ molecules per residue causes the a axis of the hexagonal unit cell to increase from 14.4 Å to 15.8 Å, with no appreciable change in the 27.0 Å c axis. Removal of the last half molecule of water results in a very diffuse α pattern, but on rehydration the sharp pattern reappears. Specimens containing five to twenty water molecules per residue show quite a different pattern, the spacing of which do not vary appreciably with hydration. This pattern includes a meridional 3.4-Å reflection, a feature commonly shown by β structures, and indeed all the reflections can be satisfactorily indexed in terms of a monoclinic unit cell with a = 9.26 Å, b = 22.05 Å, c = 6.76 Å, and γ = 108.9°. These dimensions are shown by models to be compatible with a β pleated-sheet structure.     

An X-ray diffraction study of poly-L-homoarginine hydrochloride.

An X-ray study of the synthetic polypeptide poly(L -homoarginine hydrochloride) has been made to investigate whether, like the chemically related polypeptides poly(L -lysine hydrochloride), poly(L -arginine hydrochloride), and poly(L -ornithine hydrobromide), it can undergo conformational transitions merely from variations in its degree of hydration. X-ray photographs of powder and oriented specimens containing one to 15 molecules of water per L -homoarginine hydrochloride residue showed that this polymer forms only a β-pleated-sheet structure. The pleated sheets, formed by antiparallel polypeptide chains hydrogen-bonded to each other, are piled up along the b axis in an alternating sequence (“sandwich structure”). This structure did not appreciably change with variations of the degree of hydration, and the observed reflections at 56% relative humidity (1.8 molecules of water per residue) could be indexed satisfactorily in terms of a monoclinic unit cell, of space group P2₁, with a = 9.34 Å, b = 40.07 Å, c = 6.94 Å, and γ = 106°. These dimensions are shown by models to be compatible with the proposed structure, and the calculated density of 1.27 g/cm³ agrees well with the experimental value of 1.29 g/cm³. Removal of the last molecule of water results in a very diffuse pattern, while specimens containing 20 molecules of water per residue show only reflections due to water.     

An X-ray diffraction study of alterations in bovine lung surfactant bilayer structures induced by albumin

Lung surfactant (LS) is an extra-cellular lipid-protein system responsible for maintaining low surface tension in the lung and alveolar stability. Serum proteins cause dysfunction of this material, e.g. in adult respiratory distress syndrome (ARDS). BLES is a clinically used LS consisting of most of the lipids and associated proteins from bovine lung lavage. Aqueous phases of BLES at 30% and 70% hydration, with and without 5% by weight of bovine serum albumin (BSA), calculated on the amount of lipids, were studied using X-ray diffraction during cooling from 42 to 5 degrees C. The diffraction curves are consistent with a transition from a lamellar liquid crystalline phase to a gel phase transition at cooling in the interval 30-20 degrees C. The long-spacings correspond to a reduction of the bilayer thickness during this transition. The wide-angle region shows a peak at 4.1 A below 25 degrees C, which is characteristic of the hexagonal chain packing of the gel phase. The perturbation of the bilayers by the presence of BSA seems to induce a significant decrease of the bilayer thickness. Calculations on the observed limits of swelling (taking place in the range 50-60%) indicate that BSA is closely associated with the BLES bilayers, probably due to electrostatic interaction with the cationic surfactant proteins SP-B and SP-C. This study show that the LS lipid structural organizations are extremely susceptible to small amounts of serum albumin, which may have implications in surfactant related lung disease and clinical applications of surfactant therapy.     

An X-ray diffraction and X-ray absorption spectroscopy joint study of neuroglobin

Neuroglobin (Ngb) is a member of the globin family expressed in the vertebrate brain, involved in neuroprotection. A combined approach of X-ray diffraction (XRD) on single crystal and X-ray absorption spectroscopy (XAS) in solution, allows to determine the oxidation state and the structure of the Fe-heme both in the bis-histidine and the CO-bound (NgbCO) states. The overall data demonstrate that under X-ray the iron is photoreduced fairly rapidly, and that the previously reported X-ray structure of ferric Ngb [B. Vallone, K. Nienhaus, M. Brunori, G.U. Nienhaus, Proteins 56 (2004) 85-92] very likely refers to a photoreduced species indistinguishable from the dithionite reduced protein. Results from the XAS analysis of NgbCO in solution are in good agreement with XRD data on the crystal. However prolonged X-ray exposure at 15 K determines CO release. This preliminary result paves the way to experiments aimed at the characterization of pentacoordinate ferrous Ngb, the only species competent in binding external ligands such as O₂, CO or NO.     

X-ray diffraction from membrane protein nanocrystals

Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.     

An X-ray diffraction study of poly(L-lysine hydrobromide)

A structural study of the synthetic polypeptide poly(L -lysine hydrobromide) has been made by X-ray fiber techniques. The investigation was undertaken to determine whelther this polymer undergoes conformational transitions as a function of hydration in a manner similar to other chemically related basic polypeptides. Specifically, a comparison with the previously reported structures of the hydrochloride form of poly(L -lysine) was sought. Homogeneous powder mixtures with various amounts of water and oriented fibers of poly(L -lysine hydrobromide) at different relative humidities were X-ray photographed. Reversible transitions amorphous state ? β-pleated sheet ? α-helix ? isotropic solution as a function of increasing/decreasing degrees of hydration were found. The β-pleated-sheet conformation was observed between 33% and 76% relative humidities (containing about one and three molecules of water per residue, respectively). Each pleated sheet was formed by “antiparallel” chains, and the sheets were piled up along the b-axis. The spacings of this conformation did not vary appreciably with hydration. The observed reflections at 52% relative humidity (1.4 molecules of water per residue) could be indexed satisfactorily in terms of an orthorhombic unit cell, of space group P2₁22₁, with a = 9.52 Å, b = 16.44 Å, and c = 6.80 Å. These dimensions were shown by models to be compatible with the proposed structure. The α-helix conformation was present in specimens photographed at 76% relative humidity and up, and containing between three and fifteen molecules of water per residue. The helices were packed parallel to each other in a hexagonal array but randomly along or about their lengths. Increasing the hydration from five to fifteen molecules of water per residue causes the a-axis to increase from 16.9 to 20.8 Å. Twenty molecules of water per residue produced an isotropic solution. Despite some structural differences between the hydrobromide and hydrochloride forms it is concluded that the role played by the anions is mainly related to determining the water content levels at which conformational changes occur. Therefore, the anions do not significantly influence the prevailing conformation in this particular system, but might affect the packing arrangement of the polypeptide chains.     

Influence of local anesthetics on the phosphatidylcholine model membrane: small-angle synchrotron X-ray diffraction and neutron scattering study

The phase preferences of egg yolk phosphatidylcholine (EYPC) have been examined in the presence of tertiary amine anesthetics [2-(propyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C3A) and [2-(heptyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C7A, heptacaine). Using the synchrotron small-angle X-ray diffraction (SAXD), it is shown that the C3A anesthetic induces the cubic and hexagonal (H(I)) phases at 2 > or = C3A:EYPC > 0.5 and H2O:EYPC < or = 40 molar ratios. In contrast, longer alkyloxy chain homolog C7A has no effect on the bilayer arrangement of EYPC at C7A:EYPC < = 1 molar ratios as observed by SAXD in C7A + EYPC mixtures hydrated at H2O:EYPC < = 40 molar ratios, as well as in sonicated C7A + EYPC mixtures hydrated in excess water as proved by the small-angle neutron scattering (SANS). The bilayer thickness d(L) decreases and the bilayer C7A surface area SC7A increases with the increase of C7A:EYPC molar ratio. It is suggested that the ability of tertiary amine local anesthetics to influence the dL and SC7A values and EYPC polymorphism is caused by their effective molecular shape and by charge. The possibility that anesthetic molecules may exert some of their biological effects by virtue of these properties is discussed.     

Difficult macromolecular structures determined using X-ray diffraction techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.     

Methods for X-ray diffraction analysis of macromolecular structures.

A modern approach to protein crystallography relies as much on molecular biology as on the 'core' crystallographic disciplines. Some recent, biologically significant structure determinations have demonstrated this and show the importance of new third generation synchrotron sources. Novel uses of well known phasing techniques have also been valuable in these structure determinations. For the majority of structures, advances in phasing techniques, data collection and processing and the associated computer programs have led to more effective structure determinations.     

X-ray diffraction study of cholesterol-phosphatidylserine mixtures

Phosphatidylserine-cholesterol mixtures at a molar ratio of 2:1 were investigated by X-ray diffraction. Phase separation of cholesterol independent of temperature was detected, indicating limited solubility of cholesterol in phosphatidylserine bilayers. The second phase present, the mixed phospholipid-cholesterol phase, continued to undergo melting as determined by changes with temperature in both the small angle scattering profile and in the acyl chain packing.     

X-ray diffraction study of the cornea

Low-angle X-ray diffraction patterns have been recorded from the cornea. A fibre diagram was obtained: the reflections from the axial period of collagen were on the equator while reflections from the collagen fibril lattice structure were on the meridian. Only the reflections from tha array of collagen fibrils have been studied. These reflections contain a primary first-order reflection and up to four subsidiary maxima. The first-order reflection from the array provides an estimate of the interfibril separation distance. Evidence is presented that the subsidiary maxima are consistent with the intensity transform of a uniform cylinder with a constant radius. Values for the fibril diameters and the interfibril distances are obtained for corneas from rabbit, cow and frog and from corneas of two marine fishes: toadfish and skate. Although the volume fraction of the collagen fibrils cannot be directly evaluated, an upper limit can be given. Thus, an upper limit of 0.28 was obtained for rabbit cornea.     

An ordered single-stranded structure for polyadenylic acid in denaturing solvents. An X-ray fiber diffraction and model building study

A new ordered structure has been observed in fibers of poly(rA) made from strongly denaturing solvents such as formamide or dimethylsulfoxide. The X-ray diffraction pattern together with other evidence suggests that this unique “denatured” structure is a single-stranded helix where the adenylyl residues are related by a 180 ° rotation and a 2.9 Å advance along the helix axis. The backbone is located in the center of the structure and the bases point outwards. The bases do not participate in either direct hydrogen-bonding or in stacking interactions.     

Synchrotron X-ray diffraction study of corneal stroma

Using a synchrotron X-ray source, it has been possible to record a low-angle diffraction pattern from fresh bovine corneal stroma.The pattern can be interpreted as arising from the short-range order packing of collagen fibrils in lamellae. Model calculations suggest that the positions of the fibrils remain correlated over distances corresponding to, at most, three fibril diameters (~ 120 nm). These results support theories of transparency of the cornea based on short-range order.Further, a study of the fibril spacing as a function of hydration confirms that water uptake occurs largely between the lamellae and in regions devoid of collagen fibrils, and shows that the fibril diameter increases with hydration.     

Study on crystal structures of enzyme-hydrolyzed cellulosic materials by X-ray diffraction

Different cellulosic materials were treated with different extraceller microbial enzymes. Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme and the type of cellulosic materials. Both endoglucanase and crude cellulase have pronounced effects on the structure of cellulose. The variation of crystal structure was found to attack preferentially the (0 0 2) crystal planes for cellulose I during enzymatic hydrolysis.