Toward the realization of nanoscale device control, we report a molecular valve embedded in a membrane that can be opened by illumination with long-wavelength ultraviolet (366 nanometers) light and then resealed by visible irradiation. The valve consists of a channel protein, the mechanosensitive channel of large conductance (MscL) from Escherichia coli, modified by attachment of synthetic compounds that undergo light-induced charge separation to reversibly open and close a 3-nanometer pore. The system is compatible with a classical encapsulation system, the liposome, and external photochemical control over transport through the channel is achieved.

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

This protocol details the chemical modification of the mechanosensitive channel of large-conductance (MscL) channel protein into a light-activated nanovalve and its utilization for triggered delivery in synthetic liposomal vesicles. It is based on charge-induced activation of this otherwise mechanosensitive channel by covalent attachment to the protein of rationally designed synthetic functionalities. In the dark, these functionalities will be uncharged and the channel will stay closed, but UV illumination will cause their ionization and trigger channel activity. In the case of reversible activation, subsequent illumination with visible light will neutralize the charge, causing the channel to close. The protocol includes synthesis of light-responsive compounds, protein isolation and its chemical labeling, reconstitution of the protein into artificial membranes, its analysis at the single-molecule level and its application in liposomal delivery. The whole protocol takes 4 days. Unlike mutagenesis, this method allows the introduction of custom-designed functional groups.

Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobilitymass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.ion mobility mass spectrometry | MscL | membrane proteins | structure function | ion channel gating O ne of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N-and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3-9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation-i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patchclamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13-15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach. Results and DiscussionDetergents that are Unstable in the Gas Phase Allow Detection of MscL in Its Native Form. Native mass ...