Vesicle adhesion and fusion studied by small-angle x-ray scattering.

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca2+ or Mg2+ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ∼1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis.

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins

Ambiguities and completeness of SAS data analysis: investigations of apoferritin by SAXS/SANS EID and SEC-SAXS methods

The method of small angle scattering (SAS) is widely used in the field of biophysical research of proteins in aqueous solutions. Obtaining low-resolution structure of proteins is still a highly valuable method despite the advances in high-resolution methods such as X-ray diffraction, cryo-EM etc. SAS offers the unique possibility to obtain structural information under conditions close to those of functional assays, i.e. in solution, without different additives, in the mg/mL concentration range. SAS method has a long history, but there are still many uncertainties related to data treatment. We compared 1D SAS profiles of apoferritin obtained by X-ray diffraction (XRD) and SAS methods. It is shown that SAS curves for X-ray diffraction crystallographic structure of apoferritin differ more significantly than it might be expected due to the resolution of the SAS instrument. Extrapolation to infinite dilution (EID) method does not sufficiently exclude dimerization and oligomerization effects and therefore could not guarantee total absence of dimers account in the final SAS curve. In this study, we show that EID SAXS, EID SANS and SEC-SAXS methods give complementary results and when they are used all together, it allows obtaining the most accurate results and high confidence from SAS data analysis of proteins

Deciphering Copper Coordination in the Mammalian Prion Protein Amyloidogenic Domain

Prions are pathological isoforms of the cellular prion protein that is responsible for transmissible spongiform encephalopathies (TSE). Cellular prion protein interacts with copper, Cu(II), through octarepeat and nonoctarepeat (non-OR) binding sites. The molecular details of Cu(II) coordination within the non-OR region are not well characterized yet. By the means of small angle x-ray scattering and x-ray absorption spectroscopic methods, we have investigated the effect of Cu(II) on prion protein folding and its coordination geometries when bound to the non-OR region of recombinant prion proteins (recPrP) from mammalian species considered resistant or susceptible to TSE. As the prion resistant model, we used ovine recPrP (OvPrP) carrying the protective polymorphism at residues A136, R154, and R171, whereas as TSE-susceptible models, we employed OvPrP with V136, R154, and Q171 polymorphism and bank vole recPrP. Our analysis reveals that Cu(II) affects the structural plasticity of the non-OR region, leading to a more compacted conformation. We then identified two Cu(II) coordination geometries: in the type 1 coordination observed in OvPrP at residues A136, R154, and R171, the metal is coordinated by four residues; conversely, the type 2 coordination is present in OvPrP with V136, R154, and Q171 and bank vole recPrP, where Cu(II) is coordinated by three residues and by one water molecule, making the non-OR region more exposed to the solvent. These changes in copper coordination affect the recPrP amyloid aggregation. This study may provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE

Low pH-induced conformational change and dimerization of sortilin triggers endocytosed ligand release

Sortilin is an endocytosis receptor with a luminal β-propeller domain. Here the authors present the structures of the β-propeller domain at neutral and acidic pH, which reveal that sortilin dimerises and undergoes conformational changes at low pH and further propose a model for low pH-induced ligand release by endocytosis receptors