Clustering and dynamics of phototransducer signaling domains revealed by site-directed spin labeling electron paramagnetic resonance on SRII/HtrII in membranes and nanodiscs.

In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.

Spatial organization of lipid phases in micropatterned polymer-supported membranes.

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.

Diffusion and interaction dynamics of individual membrane protein complexes confined in micropatterned polymer-supported membranes.

Micropatterned polymer-supported membranes (PSM) are established as a tool for confining the diffusion of transmembrane proteins for single molecule studies. To this end, a photochemical surface modification with hydrophobic tethers on a PEG polymer brush is implemented for capturing of lipid vesicles and subsequent fusion. Formation of contiguous membranes within micropatterns is confirmed by scanning force microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolved single-molecule tracking and localization microscopy. Free diffusion of transmembrane proteins reconstituted into micropatterned PSM is demonstrated by FRAP and by single-molecule tracking. By exploiting the confinement of diffusion within micropatterned PSM, the diffusion and interaction dynamics of individual transmembrane receptors are quantitatively resolved.

Reconstitution of membrane proteins into polymer-supported membranes for probing diffusion and interactions by single molecule techniques.

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.

Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions.

We present an ultrasensitive technique for quantitative protein-protein interaction analysis in a two-dimensional format based on phase-separated, micropatterned membranes. Interactions between proteins captured to lipid probes via an affinity tag trigger partitioning into the liquid-ordered phase, which is readily quantified by fluorescence imaging. Based on a calibration with well-defined low-affinity protein-protein interactions, equilibrium dissociation constants >1 mM were quantified. Direct capturing of proteins from mammalian cell lysates enabled us to detect homo- and heterodimerization of signal transducer and activator of transcription proteins. Using the epidermal growth factor receptor (EGFR) as a model system, quantification of low-affinity interactions between different receptor domains contributing to EGFR dimerization was achieved. By exploitation of specific features of the membrane-based assay, the regulation of EGFR dimerization by lipids was demonstrated.

Rapid transfer of transmembrane proteins for single molecule dimerization assays in polymer-supported membranes.

Dimerization of transmembrane receptors is a key regulatory factor in cellular communication, which has remained challenging to study under well-defined conditions in vitro. We developed a novel strategy to explore membrane protein interactions in a controlled lipid environment requiring minute sample quantities. By rapid transfer of transmembrane proteins from mammalian cells into polymer-supported membranes, membrane proteins could be efficiently fluorescence labeled and reconstituted with very low background. Thus, differential ligand-induced dimerization of the type I interferon (IFN) receptor subunits IFNAR1 and IFNAR2 could be probed quantitatively at physiologically relevant concentrations by single molecule imaging. These measurements clearly support a regulatory role of the affinity of IFNs toward IFNAR1 for controlling the level of receptor dimerization.