Single-molecule tracking is a powerful method to study molecular dynamics in living systems including biological membranes. High-resolution single-molecule tracking requires a bright and stable signal, which has typically been facilitated by nanoparticles due to their superb optical properties. However, there are concerns about using a nanoparticle to label a single molecule because of its relatively large size and the possibility of cross-linking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects using nanoparticles to measure the diffusion of single-membrane molecules. By conjugating a low density of streptavidin (sAv) to gold nanoparticles (AuNPs) of different sizes (10, 15, 20, 30, and 40 nm), we isolate and quantify the effect of the particle size on the diffusion of biotinylated lipids in supported lipid bilayers (SLBs). We find that single sAv tends to cross-link two biotinylated lipids, leading to a much slower diffusion in SLBs. We further demonstrate a simple and robust strategy for the monovalent and oriented labeling of a single lipid molecule with a AuNP by using naturally dimeric rhizavidin (rAv) as a bridge, thus connecting the biotinylated nanoparticle surface and biotinylated target molecule. The rAv−AuNP conjugate demonstrates fast and free diffusion in SLBs (2−3 μm 2 /s for rAv−AuNP sizes of 10−40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and cross-linking effects are successfully avoided. We also note that the diffusion of dyelabeled lipids critically depends on the choice of dye, which could report different diffusion coefficients by about 20% (2.2 μm 2 /s of ATTO647N and 2.6 μm 2 /s of ATTO532). By comparing the diffusion of the uniformly and randomly oriented labeling of a single lipid molecule with a AuNP, we conclude that oriented labeling is favorable for measuring the diffusion of single-membrane molecules. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the cross-linker for labeling. The demonstrated approach of monovalent and oriented AuNP labeling provides the opportunity to study single-molecule membrane dynamics at much higher spatiotemporal resolutions and, most importantly, without labeling artifacts.

Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer-cushioned plasma membrane bilayers that are potentially useful for the future developments of membrane-based biosensors and analytical assays.

Recent studies have suggested that the opening and closing of Hyperpolarization-activated Cyclic Nucleotide-gated 'HCN' channels involve a step that is voltage-independent, which depends upon a region that resides within the S4 and S6 transmembrane domains of the channels. In the current study, we use two electrode voltage clamp of the HCN2 channel expressed

No abstract

No abstract