Patrick Seelheim

New Synthetic Procedures to Catena-Phosphorus Cations: Preparation and Dissociation of the First cyclo-Phosphino-halophosphonium Salts

Chlorination of 1,2,3,4-tetracyclohexyl-cyclo-tetraphosphine (2) by PhICl(2) or PCl(5) in the presence of Me(3)SiOTf or GaCl(3) provides a stepwise approach to salts of the first cyclo-phosphino-chlorophosphonium cations [Cy(4)P(4)Cl](+) ([19](+)) and [Cy(4)P(4)Cl(2)](2+) ([20](2+)). The analogous iodo derivative [Cy(4)P(4)I](+) ([17](+)) is obtained as the tetraiodogallate salt from reaction of 2 with I(2) in the presence of GaI(3). Reactions of the dication [20](2+) with PMe(3) or dmpe effect a dissociation of the cyclic framework resulting in the formation of salts containing [Me(3)PPCyPCyPMe(3)](2+) ([27](2+)), [dmpeCyP](2+) ([29](2+)), and [dmpeCyPCyP](2+) ([30](2+)), respectively. The new cations represent phosphine complexes of the [PCy](2+) and [P(2)Cy(2)](2+) cationic fragments from [20](2+), demonstrating the coordinate nature of the phosphinophosphonium bonds in cyclo-phosphino-halophosphonium cations. The compounds have been characterized by NMR spectroscopy, single crystal X-ray crystallography, and Raman spectroscopy.

Layer-by-layer deposition of vesicles mediated by supramolecular interactions.

Vesicles are dynamic supramolecular structures with a bilayer membrane consisting of lipids or synthetic amphiphiles enclosing an aqueous compartment. Lipid vesicles have often been considered as mimics for biological cells. In this paper, we present a novel strategy for the preparation of three-dimensional multilayered structures in which vesicles containing amphiphilic β-cyclodextrin are interconnected by proteins using cyclodextrin guests as bifunctional linker molecules. We compared two pairs of adhesion molecules for the immobilization of vesicles: mannose-concanavalin A and biotin-streptavidin. Microcontact printing and thiol-ene click chemistry were used to prepare suitable substrates for the vesicles. Successful immobilization of intact vesicles through the mannose-concanavalin A and biotin-streptavidin motifs was verified by fluorescence microscopy imaging and dynamic light scattering, while the vesicle adlayer was characterized by quartz crystal microbalance with dissipation monitoring. In the case of the biotin-streptavidin motif, up to six layers of intact vesicles could be immobilized in a layer-by-layer fashion using supramolecular interactions. The construction of vesicle multilayers guided by noncovalent vesicle-vesicle junctions can be taken as a minimal model for artificial biological tissue.

Highly Parallel Transport Recordings on a Membrane-on-Nanopore Chip at Single Molecule Resolution

Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250 000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.

Common exonic missense variants in the C2 domain of the human KIBRA protein modify lipid binding and cognitive performance

The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer’s disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca2+ dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.

OprG Harnesses the Dynamics of its Extracellular Loops to Transport Small Amino Acids across the Outer Membrane of Pseudomonas aeruginosa.

OprG is an outer membrane protein of Pseudomonas aeruginosa whose function as an antibiotic-sensitive porin has been controversial and not well defined. Circumstantial evidence led to the proposal that OprG might transport hydrophobic compounds by using a lateral gate in the barrel wall thought to be lined by three conserved prolines. To test this hypothesis and to find the physiological substrates of OprG, we reconstituted the purified protein into liposomes and found it to facilitate the transport of small amino acids such as glycine, alanine, valine, and serine, which was confirmed by Pseudomonas growth assays. The structures of wild-type and a critical proline mutant were determined by nuclear magnetic resonance in dihexanoyl-phosphatidylcholine micellar solutions. Both proteins formed eight-stranded β-barrels with flexible extracellular loops. The interfacial prolines did not form a lateral gate in these structures, but loop 3 exhibited restricted motions in the inactive P92A mutant but not in wild-type OprG.

Reconstitution of calcium-mediated exocytosis of dense-core vesicles

Calcium control of exocytosis has been reconstituted in a hybrid system with purified DCVs and supported target membranes. Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)–dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction.

Tethered proteoliposomes containing human ABC transporter MRP3: new perspectives for biosensor application based on transmembrane proteins.

While transmembrane proteins and transporters comprise one of the largest protein families, their use in biosensors like biochips or lab-on-a-chip devices has so far been limited by their demanding requirements of a stable and compartmentalized lipid environment. A possible remedy lies in the tethering of proteoliposomes containing the reconstituted transmembrane protein to the biosensoric surface. As a proof of concept, we reconstituted the human ABC transporter MRP3 into biotinylated proteoliposomes and tethered those to a gold surface coated with streptavidin on a biotinylated self-assembled thiol monolayer. The tethering process was investigated by quartz crystal microbalance with dissipation monitoring. The final assembly of tethered proteoliposomes exhibited biological activity in terms of drug-stimulated ATP hydrolysis and substrate translocation. The presented facile immobilization approach can be easily extended to other transmembrane proteins as it does not require any modification of the protein and will open up transmembrane proteins for future application in biosensors.

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.A combination of fluorescence approaches that permit conformational changes of SNARE proteins to be visualized in different lipid environments reveals interactions underlying vesicle–membrane fusion.