Binding of interferon reduces the force of unfolding for interferon receptor 1.

Differential signaling of the type I interferon receptor (IFNAR) has been correlated with the ability of its subunit, IFNAR1, to differentially recognize a large spectrum of different ligands, which involves intricate conformational re-arrangements of multiple interacting domains. To shed light onto the structural determinants governing ligand recognition, we compared the force-induced unfolding of the IFNAR1 ectodomain when bound to interferon and when free, using the atomic force microscope and steered molecular dynamics simulations. Unexpectedly, we find that IFNAR1 is easier to mechanically unfold when bound to interferon than when free. Analysis of the structures indicated that the origin of the reduction in unfolding forces is a conformational change in IFNAR1 induced by ligand binding.

Semiconductor-Metal Nanofloret Hybrid Structures by Self-Processing Synthesis.

We present a synthetic strategy that takes advantage of the inherent asymmetry exhibited by semiconductor nanowires prepared by Au-catalyzed chemical vapor deposition (CVD). The metal-semiconductor junction is used for activating etch, deposition, and modification steps localized to the tip area using a wet-chemistry approach. The hybrid nanostructures obtained for the coinage metals Cu, Ag, and Au resemble the morphology of grass flowers, termed here Nanofloret hybrid nanostructures consisting of a high aspect ratio SiGe nanowire (NW) with a metallic nanoshell cap. The synthetic method is used to prepare hybrid nanostructures in one step by triggering a programmable cascade of events that is autonomously executed, termed self-processing synthesis. The synthesis progression was monitored by ex situ transmission electron microscopy (TEM), in situ scanning transmission electron microscopy (STEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses to study the mechanistic reaction details of the various processes taking place during the synthesis. Our results indicate that the synthesis involves distinct processing steps including localized oxide etch, metal deposition, and process termination. Control over the deposition and etching processes is demonstrated by several parameters: (i) etchant concentration (water), (ii) SiGe alloy composition, (iii) reducing agent, (iv) metal redox potential, and (v) addition of surfactants for controlling the deposited metal grain size. The NF structures exhibit broad plasmonic absorption that is utilized for demonstrating surface-enhanced Raman scattering (SERS) of thiophenol monolayer. The new type of nanostructures feature a metallic nanoshell directly coupled to the crystalline semiconductor NW showing broad plasmonic absorption.

Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane.

Interactions of proteins in the plasma membrane are notoriously challenging to study under physiological conditions. We report in this paper a generic approach for spatial organization of plasma membrane proteins into micropatterns as a tool for visualizing and quantifying interactions with extracellular, intracellular, and transmembrane proteins in live cells. Based on a protein-repellent poly(ethylene glycol) polymer brush, micropatterned surface functionalization with the HaloTag ligand for capturing HaloTag fusion proteins and RGD peptides promoting cell adhesion was devised. Efficient micropatterning of the type I interferon (IFN) receptor subunit IFNAR2 fused to the HaloTag was achieved, and highly specific IFN binding to the receptor was detected. The dynamics of this interaction could be quantified on the single molecule level, and IFN-induced receptor dimerization in micropatterns could be monitored. Assembly of active signaling complexes was confirmed by immunostaining of phosphorylated Janus family kinases, and the interaction dynamics of cytosolic effector proteins recruited to the receptor complex were unambiguously quantified by fluorescence recovery after photobleaching.

Monolayer contact doping of silicon surfaces and nanowires using organophosphorus compounds.

Monolayer Contact Doping (MLCD) is a simple method for doping of surfaces and nanostructures(1). MLCD results in the formation of highly controlled, ultra shallow and sharp doping profiles at the nanometer scale. In MLCD process the dopant source is a monolayer containing dopant atoms. In this article a detailed procedure for surface doping of silicon substrate as well as silicon nanowires is demonstrated. Phosphorus dopant source was formed using tetraethyl methylenediphosphonate monolayer on a silicon substrate. This monolayer containing substrate was brought to contact with a pristine intrinsic silicon target substrate and annealed while in contact. Sheet resistance of the target substrate was measured using 4 point probe. Intrinsic silicon nanowires were synthesized by chemical vapor deposition (CVD) process using a vapor-liquid-solid (VLS) mechanism; gold nanoparticles were used as catalyst for nanowire growth. The nanowires were suspended in ethanol by mild sonication. This suspension was used to dropcast the nanowires on silicon substrate with a silicon nitride dielectric top layer. These nanowires were doped with phosphorus in similar manner as used for the intrinsic silicon wafer. Standard photolithography process was used to fabricate metal electrodes for the formation of nanowire based field effect transistor (NW-FET). The electrical properties of a representative nanowire device were measured by a semiconductor device analyzer and a probe station.

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.

Maleimide photolithography for single-molecule protein-protein interaction analysis in micropatterns.

Spatial organization of proteins into microscopic structures has important applications in fundamental and applied research. Preserving the function of proteins in such microstructures requires generic methods for site-specific capturing through affinity handles. Here, we present a versatile bottom-up surface micropatterning approach based on surface functionalization with maleimides, which selectively react with organic thiols. Upon UV irradiation through a photomask, the functionality of illuminated maleimide groups was efficiently destroyed. Remaining maleimides in nonilluminated regions were further reacted with different thiol-functionalized groups for site-specific protein immobilization under physiological conditions. Highly selective immobilization of His-tagged proteins into tris(nitrilotriacetic acid) functionalized microstructures with very high contrast was possible even by direct capturing of proteins from crude cell lysates. Moreover, we employed phosphopantetheinyl transfer from surface-immobilized coenzyme A to ybbR-tagged proteins in order to implement site-specific, covalent protein immobilization into microstructures. The functional integrity of the immobilized protein was confirmed by monitoring protein-protein interactions in real time. Moreover, we demonstrate quantitative single-molecule analysis of protein-protein interactions with proteins selectively captured into these high-contrast micropatterns.

Functional immobilization and patterning of proteins by an enzymatic transfer reaction.

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.