An amyloid form of the protein α-synuclein is the major component of the intraneuronal inclusions called Lewy bodies, which are the neuropathological hallmark of Parkinson's disease (PD). α-Synuclein is known to associate with anionic lipid membranes, and interactions between aggregating α-synuclein and cellular membranes are thought to be important for PD pathology. We have studied the molecular determinants for adsorption of monomeric α-synuclein to planar model lipid membranes composed of zwitterionic phosphatidylcholine alone or in a mixture with anionic phosphatidylserine (relevant for plasma membranes) or anionic cardiolipin (relevant for mitochondrial membranes). We studied the adsorption of the protein to supported bilayers, the position of the protein within and outside the bilayer, and structural changes in the model membranes using two complementary techniquesquartz crystal microbalance with dissipation monitoring, and neutron reflectometry. We found that the interaction and adsorbed conformation depend on membrane charge, protein charge, and electrostatic screening. The results imply that α-synuclein adsorbs in the headgroup region of anionic lipid bilayers with extensions into the bulk but does not penetrate deeply into or across the hydrophobic acyl chain region. The adsorption to anionic bilayers leads to a small perturbation of the acyl chain packing that is independent of anionic headgroup identity. We also explored the effect of changing the area per headgroup in the lipid bilayer by comparing model systems with different degrees of acyl chain saturation. An increase in area per lipid headgroup leads to an increase in the level of α-synuclein adsorption with a reduced water content in the acyl chain layer. In conclusion, the association of α-synuclein to membranes and its adsorbed conformation are of electrostatic origin, combined with van der Waals interactions, but with a very weak correlation to the molecular structure of the anionic lipid headgroup. The perturbation of the acyl chain packing upon monomeric protein adsorption favors association with unsaturated phospholipids preferentially found in the neuronal membrane.

Group A rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 were detected by enzyme immunoassay or reverse transcription-PCR in 61, 14, 6, and 3% of stool specimens from 414 children consulting for gastroenteritis between 1995 and 1998. These data highlight the importance of caliciviruses in infantile gastroenteritis. Among these, Norwalk-like viruses belonging to genogroup II were predominant.

Neutron crystallography is an important complementary technique to X-ray crystallography because it provides details of the H-atom and proton (H+) positions in biological macromolecules and, given the absence of radiation damage with neutrons, the resulting structures are 'damage-free' even at room temperature. Knowledge of the positions of the H-atoms and protons is important since details of protonation and hydration are often necessary for understanding macromolecular function at an atomic level, such as enzyme mechanisms [1,] and in drug-binding [2,]. Although, historically, the study of biological macromolecules using neutron crystallography had been limited-due to the requirement for extremely large crystals of several cubic millimetres-recent improvements to the quasi-Laue diffractometer LADI-III at the Insitut Laue-Langevin (ILL) are allowing us to extend the limits for neutron macromolecular crystallography using crystals of smaller dimensions and studying larger unit-cell systems [3]. This has resulted in a typical lower limit for useful crystal volumes of ~0.1 mm3, however, from a very recent study of human recombinant insulin it has been shown that in fact much smaller crystals can be used for high-resolution neutron diffraction studies. Neutron quasi-Laue diffraction data have been collected to 2.2 Å resolution from a crystal of human insulin with a radically small volume of 0.02-0.03 mm3 (~250-300 μm on edge) using the LADI-III instrument at the ILL. Given that the insulin crystal used for data collection was H/D-exchanged this implies that the limit for perdeuterated crystals (in which all H are replaced by its isotope deuterium) should be even smaller, possibly less than 0.01 mm3. Given that crystal volumes of this order are much more feasible to grow, a large number of potential neutron crystallography studies are now within range. Herein we will describe, in detail, the neutron structure of human recombinant insulin, which has not been determined previously using neutron diffraction, and which clearly shows details of protonation and hydration that are not attainable even with ultra-high resolution X-ray crystallography.

Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH-) and the proton-shuttling residue His64. This distance (approximately 7.5 A) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (approximately 1.0 A) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 A resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.

Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 A. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O(2) via a mechanism that is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 A, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.