Jens B. Simonsen

Elliptical Structure of Phospholipid Bilayer Nanodiscs Encapsulated by Scaffold Proteins: Casting the Roles of the Lipids and the Protein

Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disc-like nanostructures, allowing for unprecedented solution studies of the assembly, structure, and function of membrane proteins (Bayburt et al. Nano Lett. 2002, 2, 853-856). Based on small-angle neutron scattering in combination with variable-temperature studies of synchrotron small-angle X-ray scattering on nanodiscs in solution, we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, possesses intrinsically an elliptical shape. The temperature dependence of the curvature of the nanodiscs prepared with two different phospholipid types (DLPC and POPC) shows that it is the scaffold protein that determines the overall elliptical shape and that the nanodiscs become more circular with increasing temperature. Our data also show that the hydrophobic bilayer thickness is, to a large extent, dictated by the scaffolding protein and adjusted to minimize the hydrophobic mismatch between protein and phospholipid. Our conclusions result from a new comprehensive and molecular-based model of the nanodisc structure and the use of this to analyze the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs.

Small-angle scattering gives direct structural information about a membrane protein inside a lipid environment.

Monomeric bacteriorhodopsin (bR) reconstituted into POPC/POPG-containing nanodiscs was investigated by combined small-angle neutron and X-ray scattering. A novel hybrid approach to small-angle scattering data analysis was developed. In combination, these provided direct structural insight into membrane-protein localization in the nanodisc and into the protein-lipid interactions. It was found that bR is laterally decentred in the plane of the disc and is slightly tilted in the phospholipid bilayer. The thickness of the bilayer is reduced in response to the incorporation of bR. The observed tilt of bR is in good accordance with previously performed theoretical predictions and computer simulations based on the bR crystal structure. The result is a significant and essential step on the way to developing a general small-angle scattering-based method for determining the low-resolution structures of membrane proteins in physiologically relevant environments.

Columnar Self-Assembly and Alignment of Planar Carbenium Ions in Langmuir-Blodgett Films

Structural and optical properties of multilayer Langmuir-Blodgett (LB) films of two amphiphilic carbenium salts 2-didecylamino-6,10-bis(dimethylamino)-4,8,12-trioxatriangulenium hexafluorophosphate (ATOTA-1) and 2,6-bis(decylmethylamino)-10-dimethylamino-4,8,12-trioxatriangulenium hexafluorophosphate (ATOTA-2) are described. The LB films were prepared on lipophilic glass by standard vertical dipping. Grazing incidence X-ray diffraction (GIXD) measurements show that the planar organic cores, in spite of their positive charge, form closely packed columns with a repeating distance of ∼3.45 Å. Specular X-ray reflectivity (SXR) reveals the LB multilayers to consist of Y-type bilayers with thickness 31 Å for ATOTA-1 and 41 Å for ATOTA-2. This significant difference is ascribed to the different packing motifs of the alkyl chains in the two LB films. GIXD and polarized UV-vis absorption and emission spectroscopy show that the columnar aggregates in the LB films are oriented along the dipping direction. This alignment is attributed to shear effects during LB transfer. The main absorption band of the LB films is blue-shifted compared to that in solution, while the fluorescence is red-shifted by more than 100 nm. These findings suggest the presence of H-aggregates in agreement with the cofacial packing derived from the X-ray measurements. Polarized absorption spectroscopy with variable angle of incidence was used to resolve two perpendicular optical transitions in the visible range, one at 460 nm polarized perpendicular to the columnar direction, in the plane of the film, and one at 420 nm polarized along the film normal.

Camel and Bovine Chymosin: The Relationship between Their Structures and Cheese-Making Properties.

Analysis of the crystal structures of the two milk-clotting enzymes bovine and camel chymosin has revealed that the better milk-clotting activity towards bovine milk of camel chymosin compared with bovine chymosin is related to variations in their surface charges and their substrate-binding clefts.

Close Columnar Packing of Triangulenium Ions in Langmuir Films

Three new tris(dialkylamino)trioxatriangulenium (ATOTA+) salts rendered amphiphilic by attachment of two (5a x PF6 and 5b x PF6) or four (5c x PF6) n-decyl chains have been synthesized, and their Langmuir films have been studied by grazing incidence X-ray diffraction (GIXD). Compounds 5a x PF6 and 5b x PF6 both self-assemble into 2D-crystalline Langmuir monolayers, in which the planar triangular shaped carbenium ions form columnar aggregates segregated from the PF6- ions. The column width is found to be close to the width of the triangulenium moiety itself (approximately 17 angstroms), while the repeat distance along the columnar aggregates is only 3.45 angstroms, implicating a near cofacial columnar structure with only a small tilt of the planar carbenium ions relative to the columnar axis. A detailed Bragg rod analysis confirmed an 8-9 degrees tilt and inferred a large anisotropy in the smearing/thermal displacement along the pi-pi stacking and lamellar packing directions. Specular X-ray reflectivity (SXR) was used to confirm the model derived from the GIXD data and elucidate the average position of the disordered PF6- ions, showing that the majority of the anions are accommodated in the ATOTA+ layer rather than in the water subphase.

Counterions Control Whether Self-Assembly Leads to Formation of Stable and Well-Defined Unilamellar Nanotubes or Nanoribbons and Nanorods

Self-assembly of the amphiphilic π-conjugated carbenium ion ATOTA-1(+) in aqueous solution selectively leads to discrete and highly stable nanotubes or nanoribbons and nanorods, depending on the nature of the counterion (Cl(-) vs. PF6(-), respectively). The nanotubes formed by the Cl(-) salt illustrate an exceptional example of a structural well-defined (29±2 nm in outer diameter) unilamellar tubular morphology featuring π-conjugated functionality and high stability and flexibility, in aqueous solution.

The effect of using binary mixtures of zwitterionic and charged lipids on nanodisc formation and stability

Nanodiscs are self-assembled ∼10 nm particles composed of lipid bilayer patches, stabilized by helical amphipathic belt proteins. The size, monodispersity and well-defined structure make the nanodiscs a popular model for the biological cell membrane, especially for structural and functional studies of membrane proteins. The structures and properties of nanodiscs made of zwitterionic lipids are well known. However, the biological cell membrane is negatively charged and thus nanodiscs containing anionic lipids should provide a better mimic of the native environment for membrane proteins. Despite the broad potential of charged nanodiscs, a systematic study of the influence of charged lipids on the nanodisc structure and stability has not yet been accomplished. In this paper, binary systems of zwitterionic DMPC mixed with the anionic lipids DMPG or DMPA or with the cationic synthetic DMTAP are used to prepare negatively and positively charged nanodiscs, respectively. Size exclusion chromatography analysis shows that nanodiscs can be prepared with high yield at all compositions of DMPC and DMPG, while mixtures of DMPC with either DMPA or DMTAP impair nanodisc formation. The presence of DMPG improves the stability of the nanodisc, both thermally and over time upon storage at −20 °C, as compared to pure DMPC nanodiscs. This stabilization is attributed to favourable electrostatic interactions between the anionic head of DMPG and cationic charges of the belt protein and inter-nanodisc repulsion that prevents aggregation of nanodiscs. In contrast, even small fractions of DMPA result in a faster degradation at −20 °C. These results suggest that the mixing of DMPC and DMPG provides nanodiscs that are better suited for studies of the function and structure of membrane proteins not only due to their inherent charge but also due to their improved thermal and storage stability compared to pure DMPC nanodiscs.

Structure of Dimeric and Tetrameric Complexes of the BAR Domain Protein PICK1 Determined by Small-Angle X-Ray Scattering

PICK1 is a neuronal scaffolding protein containing a PDZ domain and an auto-inhibited BAR domain. BAR domains are membrane-sculpting protein modules generating membrane curvature and promoting membrane fission. Previous data suggest that BAR domains are organized in lattice-like arrangements when stabilizing membranes but little is known about structural organization of BAR domains in solution. Through a small-angle X-ray scattering (SAXS) analysis, we determine the structure of dimeric and tetrameric complexes of PICK1 in solution. SAXS and biochemical data reveal a strong propensity of PICK1 to form higher-order structures, and SAXS analysis suggests an offset, parallel mode of BAR-BAR oligomerization. Furthermore, unlike accessory domains in other BAR domain proteins, the positioning of the PDZ domains is flexible, enabling PICK1 to perform long-range, dynamic scaffolding of membrane-associated proteins. Together with functional data, these structural findings are compatible with a model in which oligomerization governs auto-inhibition of BAR domain function.

Self-Assembly and Near Perfect Macroscopic Alignment of Fluorescent Triangulenium Salt in Spin-Cast Thin Films on PTFE

Highly fluorescent, discotic trioxatriangulenium dyes were aligned by simple spin-casting on substrates with friction transferred PTFE layers. The fluorescent crystalline thin films show near perfect macroscopic alignment on centimeter large areas directly from spin-casting. Gracing Incidence X-ray Diffraction (GIXD) unambiguously allowed the determination of a long-range order unit cell as well as its orientation with respect to the PTFE fibers. Further analysis of the X-ray data, in conjunction with polarized absorption spectroscopy, suggest a lamellar packing model with alternating layers of alkyl chains and ionic dyes oriented parallel to the substrate. This structure results in a highly anisotropic electrostatic potential around the cationic chromophore, causing significant shifts in energy and orientation of the optical transitions. Thus, the optical properties of the material are, to a large extent, controlled by the position of the otherwise inert PF6(-) counterions. The bright fluorescence from the films is also polarized parallel to the PTFE alignment layer. Doping of the thin films with fluorescent energy acceptor traps shows that efficient exciton migration takes place in the thin films. The excellent exciton transfer capabilities, in conjunction with the perfect alignment, might be of interest in future applications in solar energy harvesting or as thin film sensors.

Gene Transfer of Apolipoprotein A-V Improves the Hypertriglyceridemic Phenotype of apoa5 (−/−) mice

Objective—Apolipoprotein (apo) A-V is a low abundance protein with a profound influence on plasma triacylglycerol levels. In human populations, single nucleotide polymorphisms and mutations in APOA5 positively correlate with hypertriglyceridemia. As an approach to preventing the deleterious effects of chronic hypertriglyceridemia, apoA-V gene therapy has been pursued. Methods and Results—Recombinant adeno-associated virus (AAV) 2/8 harboring the coding sequence for human apoA-V or a control AAV2/8 was transduced into hypertriglyceridemic apoa5 (−/−) mice. After injection of 1×1012 viral genome AAV2/8-apoA-V, maximal plasma levels of apoA-V protein were achieved at 3 to 4 weeks, after which the concentration slowly declined. Complementing the appearance of apoA-V was a decrease (50±6%) in plasma triacylglycerol content compared with apoa5 (−/−) mice treated with AAV2/8-&bgr;-galactosidase. After 8 weeks the mice were euthanized and plasma lipoproteins separated. AAV2/8-apoA-V–transduced mice displayed a dramatic reduction in very low–density lipoprotein triacylglycerol content. Vector generated apoA-V in plasma associated with both very low–density lipoprotein and high-density lipoprotein fractions. Conclusion—Taken together, the data show that gene transfer of apoA-V improves the severe hypertriglyceridemia phenotype of apoa5 (−/−) mice. Given the prevalence of hypertriglyceridemia, apoA-V gene therapy offers a potential strategy for maintenance of plasma triacylglycerol homeostasis.