Cation redistribution upon dehydration of Na58Y faujasite zeolite: a joint neutron diffraction and molecular simulation study

Using neutron scattering and Monte Carlo simulation, we investigate the distribution of cations in Na₅₈Y faujasite upon (de)hydration. We introduce a new method for the assignment of cations to specific sites in molecular simulations from their local environment. This allows us to bypass the need of the coordinates of crystallographic sites, which vary as water adsorption induces changes in the zeolite framework structure. Although the agreement between experiments and simulation is excellent at high temperature, some differences are observed below 150°C. We show that these differences are due to the presence of water and that temperature itself as well as adsorption-induced deformation of the framework play a less important role. We demonstrate the migration of sodium to sites III upon water adsorption, not observed for other Si:Al ratios.     

A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data

The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X‐ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low‐resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two‐dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low‐resolution phase information that can be refined further by the conventional methods of protein crystallography.     

Exploiting powder X-ray diffraction for direct structure determination in structural biology: the P2X4 receptor trafficking motif YEQGL

We report the crystal structure of the 5-residue peptide acetyl-YEQGL-amide, determined directly from powder X-ray diffraction data recorded on a conventional laboratory X-ray powder diffractometer. The YEQGL motif has a known biological role, as a trafficking motif in the C-terminus of mammalian P2X4 receptors. Comparison of the crystal structure of acetyl-YEQGL-amide determined here and that of a complex formed with the μ2 subunit of the clathrin adaptor protein complex AP2 reported previously, reveals differences in conformational properties, although there are nevertheless similarities concerning aspects of the hydrogen-bonding arrangement and the hydrophobic environment of the leucine sidechain. Our results demonstrate the potential for exploiting modern powder X-ray diffraction methodology to achieve complete structure determination of materials of biological interest that do not crystallize as single crystals of suitable size and quality for single-crystal X-ray diffraction.     

The fibrils of Ure2p homologs from Saccharomyces cerevisiae and Saccharoymyces paradoxus have similar cross-β structure in both dried and hydrated forms

The ability to convert into amyloid fibrils is a common feature of prion proteins. However, not all amyloid-forming proteins act as prions. Here, we compared two homologs of the yeast prion protein Ure2 from Saccharomyces cerevisiae and Saccharomyces paradoxus, ScUre2p and SpUre2p, which have different prion propensities in vivo. We also addressed the controversial issue of whether hydrated fibrils of Ure2 show a fundamentally different X-ray diffraction pattern than dried samples. Using Fourier transform infrared spectrometry (FTIR) and wide angle X-ray scattering of dried and concentrated hydrated fibrils, we compared the fibril structure of ScUre2p and SpUre2p. The results show that fibrils of ScUre2p and SpUre2 have a similar cross-β core under dried and hydrated conditions, with the same inter-strand and inter-sheet spacings. Given the different prion propensity of the two Ure2p homologs, this suggests that the detailed organization of the cross-β core may play an important role in the efficiency of prion propagation.     

The 5A structure of heterologously expressed plant aquaporin SoPIP2;1

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.     

Crystal structure of a carbohydrate induced homodimer of phospholipase A2 from Bungarus caeruleus at 2.1A resolution

This is the first crystal structure of a carbohydrate induced dimer of phospholipase A(2) (PLA(2)). This is an endogenous complex formed between two PLA(2) molecules and two mannoses. It was isolated from Krait venom (Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA(2) was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0-2.1A. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA(2) molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA(2) molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.     

All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin

The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7A and 2.2 A, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hyroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with product, coleneteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.     

Crystal lattice as biological phenotype for insect viruses

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting viruses.     

Poly-(L-alanine) expansions form core beta-sheets that nucleate amyloid assembly

Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.     

Hydration properties of N-(alpha-hydroxyacyl)-sphingosine: X-ray powder diffraction and FT-Raman spectroscopic studies

The thermotropic properties of N-(alpha-hydroxyacyl)-sphingosine (CER[AS]) in dry and hydrated state were studied by means of X-ray powder diffraction and FT-Raman spectroscopy. The polymorphic states of the CER[AS]/water mixture (lamellar crystalline, lamellar hexagonal gel, liquid crystalline) depend on the thermal pre-treatment of the sample. Only by heating the CER[AS]/water mixture above the melting chain transition can the system be hydrated. At room temperature, both dry and hydrated states form lamellar structures, which differ in their repeat distance and packing of hydrocarbon chains. Above the melting chain transition, hydrated CER[AS] forms a liquid crystalline hexagonal phase, whereas anhydrous CER[AS] forms an isotropic liquid phase. The various phases of hydrated CER[AS] are distinguished on the basis of the corresponding Raman spectra.