RESUMEN
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.
Asunto(s)
Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Electricidad Estática , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Adsorción , Amiloidosis/patología , Amiloidosis/fisiopatología , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cuerpos de Lewy/química , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Lípidos de la Membrana/fisiología , Neuronas/química , Neuronas/metabolismo , Neuronas/fisiología , Difracción de Neutrones , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Fosfolípidos/química , Fosfolípidos/metabolismo , Fosfolípidos/fisiología , alfa-Sinucleína/fisiologíaRESUMEN
Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane.
Asunto(s)
Toxina Diftérica , Membrana Dobles de Lípidos/metabolismo , Estructura Terciaria de Proteína , Membrana Celular/metabolismo , Toxina Diftérica/química , Toxina Diftérica/metabolismo , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Modelos Moleculares , Neutrones , Resonancia Magnética Nuclear BiomolecularRESUMEN
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.
Asunto(s)
Anhidrasa Carbónica II/química , Sitios de Unión , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Anhidrasa Carbónica II/biosíntesis , Anhidrasa Carbónica II/genética , Cristalografía por Rayos X , Deuterio , Humanos , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
Alcanfor 5-Monooxigenasa/biosíntesis , Alcanfor 5-Monooxigenasa/química , Alcanfor 5-Monooxigenasa/genética , Cristalización , Cristalografía por Rayos X , Deuterio , Escherichia coli/química , Hidrógeno/química , Marcaje Isotópico , Modelos Moleculares , Neutrones , Conformación Proteica , Pseudomonas putida/enzimología , Agua/químicaRESUMEN
Yeast inorganic pyrophosphatase (Y-PPase) is a model system for studying phosphoryl-transfer reactions catalysed by multiple metal ions. To understand the process requires knowledge of the positions of the protons in the active site, which can be best achieved by neutron diffraction analysis. In order to reduce the hydrogen incoherent-scattering background and to improve the signal-to-noise ratio of the neutron reflections, deuterated protein was produced. Deuterated protein 96% enriched with deuterium was produced in high yield and crystals as large as 2 mm on one side were obtained. These crystals have unit-cell parameters a = 58.9, b = 103.9, c = 117.0 A, alpha = beta = gamma = 90 degrees at 273 K and diffract neutrons to resolutions of 2.5-3 A. The X-ray structure of the perdeuterated protein has also been refined at 273 K to 1.9 A resolution.