Modeling Detergent Organization around Aquaporin-0 Using Small-Angle X-ray Scattering

Solubilization of integral membrane proteins in aqueous solutions requires the presence of amphiphilic molecules like detergents. The transmembrane region of the proteins is then surrounded by a corona formed by these molecules, ensuring a hydrophilic outer surface. The presence of this corona has strongly hampered structural studies of solubilized membrane proteins by small-angle X-ray scattering (SAXS), a technique frequently used to monitor conformational changes of soluble proteins. Through the online combination of size exclusion chromatography, SAXS, and refractometry, we have determined a precise geometrical model of the <i>n</i>-dodecyl β-d-maltopyranoside corona surrounding aquaporin-0, the most abundant membrane protein of the eye lens. The SAXS data were well-fitted by a detergent corona shaped in an elliptical toroid around the crystal structure of the protein, similar to the elliptical shape recently reported for nanodiscs (Skar-Gislinge et al. <i>J. Am. Chem. Soc.</i><b>2010</b>, <i>132</i>, 13713–13722). The torus thickness determined from the curve-fitting protocol is in excellent agreement with the thickness of a lipid bilayer, while the number of detergent molecules deduced from the volume of the torus compares well with those obtained on the same sample from refractometry and mass analysis based on SAXS forward scattering. For the first time, the partial specific volume of the detergent surrounding a protein was measured. The present protocol is a crucial step toward future conformational studies of membrane proteins in solution

Ab Initio and All-Atom Modeling of Detergent Organization around Aquaporin‑0 Based on SAXS Data

A necessary initial step for the application of small angle X-ray scattering (SAXS) as an analytical probe for structural investigations of membrane proteins in solution is the precise knowledge of the structure of spontaneously formed detergent assemblies around the protein. Following our recent article (Berthaud et al. <i>J. Am. Chem. Soc.</i> <b>2012</b>, <i>134</i>, 10080–10088) on the study of the n-dodecyl β-d-maltopyranoside (dDM) corona surrounding Aquaporin-0 tetramers in solution, we aimed at the development of more elaborate models, exploiting the information content of the scattering data. Two additional approaches are developed here for the fit of SAXS experimental data, one based on a generalized ab initio algorithm for the construction of a coarse-grained representation of the detergent assemblies, and a second based on atomistic molecular dynamics. Accordingly, we are able to fit the SAXS experimental data and obtain a better insight concerning the structure of the detergent corona around the hydrophobic part of the Aquaporin-0 surface. The present analysis scheme represents an additional step toward future conformational studies of transmembrane proteins in solution

Topological Connection between Vesicles and Nanotubes in Single-Molecule Lipid Membranes Driven by Head–Tail Interactions

Lipid nanotube–vesicle networks are important channels for intercellular communication and transport of matter. Experimentally observed in neighboring mammalian cells but also reproduced in model membrane systems, a broad consensus exists on their formation and stability. Lipid membranes must be composed of at least two molecular components, each stabilizing low (generally a phospholipid) and high curvatures. Strong anisotropy or enhanced conical shape of the second amphiphile is crucial for the formation of nanotunnels. Anisotropic driving forces generally favor nanotube protrusions from vesicles. In this work, we report the unique case of topologically connected nanotubes–vesicles obtained in the absence of directional forces, in single-molecule membranes, composed of an anisotropic bolaform glucolipid, above its melting temperature, Tm. Cryo-TEM and fluorescence confocal microscopy show the interconnection between vesicles and nanotubes in a single-phase region, between 60 and 90 °C under diluted conditions. Solid-state NMR demonstrates that the glucolipid can assume two distinct configurations, head–head and head–tail. These arrangements, seemingly of comparable energy above the Tm, could explain the existence and stability of the topologically connected vesicles and nanotubes, which are generally not observed for classical single-molecule phospholipid-based membranes above their Tm

Aggregation of the Salivary Proline-Rich Protein IB5 in the Presence of the Tannin EgCG

In the mouth, proline-rich proteins (PRP), which are major components of stimulated saliva, interact with tannins contained in food. We report in vitro interactions of the tannin epigallocatechin gallate (EgCG), with a basic salivary PRP, IB5, studied through electrospray ionization mass spectrometry (ESI-MS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS). In dilute protein (IB5) solutions of low ionic strength (1 mM), the proteins repel each other, and the tannins bind to nonaggregated proteins. ESI-MS experiments determine the populations of nonaggregated proteins that have bound various numbers of tannin molecules. These populations match approximately the Poisson distribution for binding to <i>n</i> = 8 sites on the protein. MS/MS experiments confirm that complexes containing <i>n</i> = 1 to 8 EgCG molecules are dissociated with the same energy. Assuming that the 8 sites are equivalent, we calculate a binding isotherm, with a binding free energy Δμ = 7.26<i>RT</i>_a (<i>K</i>_d = 706 μM). In protein solutions that are more concentrated (0.21 mM) and at higher ionic strength (50 mM, pH 5.5), the tannins can bridge the proteins together. DLS experiments measure the number of proteins per aggregate. This number rises rapidly when the EgCG concentration exceeds a threshold (0.2 mM EgCG for 0.21 mM of IB5). SAXS experiments indicate that the aggregates have a core–corona structure. The core contains proteins that have bound at least 3 tannins and the corona has proteins with fewer bound tannins. These aggregates coexist with nonaggregated proteins. Increasing the tannin concentration beyond the threshold causes the transfer of proteins to the aggregates and a fast rise of the number of proteins per aggregate. A poisoned growth model explains this fast rise. Very large cationic aggregates, containing up to 10 000 proteins, are formed at tannin concentrations (2 mM) slightly above the aggregation threshold (0.2 mM)

Simultaneous Phase Transfer and Surface Modification of TiO₂ Nanoparticles Using Alkylphosphonic Acids: Optimization and Structure of the Organosols

An original protocol of simultaneous surface modification and transfer from aqueous to organic phases of anatase TiO₂ nanoparticles (NPs) using alkylphosphonic acids (PAs) is studied. The influence of the solvent, the nature and concentration of the PA, and the size, concentration, and aggregation state of the TiO₂ NPs was investigated. Complete transfer was observed for linear alkyl chains (5, 8, 12, and 18 C atoms), even at very high sol concentrations. After transfer, the grafted NPs were characterized by ³¹P solid-state MAS NMR. The dispersion state of NPs before and after phase transfer was monitored by dynamic light scattering (DLS). Small-angle neutron scattering (SANS) was used to characterize the structure of PA-grafted NPs in the organic solvent. Using a quantitative core–shell model cross-checked under different contrast conditions, it is found that the primary particles making up the NPs are homogeneously grafted with a solvated PA-layer. The nanometric thickness of the latter is shown to increase with the length of the linear carbon chain of the PA, independent of the size of the primary TiO₂ NP. Interestingly, a reversible temperature-dependent aggregation was evidenced visually for C₁₈PA, and confirmed by DLS and SANS: heating the sample induces the breakup of aggregates, which reassemble upon cooling. Finally, in the case of NPs agglomerated by playing with the pH or the salt concentration of the sols, the phase transfer with PA is capable of redispersing the agglomerates. This new and highly versatile method of NP surface modification with PAs and simultaneous transfer is thus well suited for obtaining well-dispersed grafted NPs