We have established a robust and versatile analytical platform for probing membrane protein function in a defined lipid environment on solid supports. This approach is based on vesicle capturing onto an ultrathin poly(ethylene glycol) (PEG) polymer brush functionalized with fatty acid moieties and subsequent vesicle fusion into a contiguous membrane. In order to ensure efficient formation of these tethered polymer-supported membranes (PSM), very small unilamellar vesicles (VSUV) containing fluorescent lipids or model transmembrane proteins were generated by detergent depletion with cyclodextrin. Thus, very rapid reconstitution of membrane proteins into PSM was possible in a format compatible with microfluidics. Moreover, surfaces could be regenerated with detergent solution and reused multiple times. Lipid and protein diffusion in these membranes was investigated by fluorescence recovery after photobleaching, single molecule tracking, and fluorescence correlation spectroscopy. Full mobility of lipids and a high degree of protein mobility as well as homogeneous diffusion of both were observed. Quantitative ligand binding studies by solid phase detection techniques confirmed functional integrity of a transmembrane receptor reconstituted into these PSM. Colocomotion of individual ligand-receptor complexes was detected, demonstrating the applicability for single molecule fluorescence techniques.

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2 Å resolution. Relative to recent Argonaute structures, a 24°reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.RNA interference, an ancient mechanism of gene silencing mediated by small RNAs, is widely distributed throughout most eukaryotes (1, 2). Potent silencing of genes is achieved through the processing of double-stranded RNA (dsRNA) 2 into small interfering RNAs (siRNA) by the enzyme Dicer. The ϳ22-ntlong siRNAs contain a signature 5Ј-phosphate group and a 2-nt-long 3Ј-overhang (3). The siRNA is then loaded into the RNA-induced silencing complex (RISC), of which Argonaute is the primary catalytic component (4, 5). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (6 -8). Argonaute cleaves the passenger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (9 -11). This single-stranded guide strand siRNA bound to Argonaute is required for the sequence-dependent recognition of mRNA by RISC. Recombinantly expressed human Argonaute 2 in complex with single-stranded siRNA forms a minimal RISC, capable of mRNA recognition and cleavage (12). Recent crystal structures of Argonaute apoenzymes show the architecture of Argonaute to be a multidomain protein composed of N-terminal, PAZ, MID, and PIWI domains (13,14).The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conformational changes during the cycle of mRNA recognition by RISC (15, 16). Nevertheless, it remains unclear how the multidomain arrangement of Argonaute recognizes and distinguishes between singlestranded and double-stranded oligonucleotides that correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand/mRNA duplex.We report the crystal structure of Aquifex aeolicus Argonaute (AaAgo), crystallized in the presence of a single-stranded siRNA and refined using data to 3.2 Å resolution. We propose a model for Argonaute activity in the context of the RISC complex. We also ...

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand–receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-sn-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l(d)) and liquid-ordered (l(o)) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l(o) domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l(o) and l(d) phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l(d) phase. Hence, the l(o) phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains.