It is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids’ collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer.
Light can effectively interrogate biological systems providing control over complex cellular processes. Particularly advantageous features of photo-induced processes are reversibility, physiological compatibility, and spatiotemporal precision. Understanding the underlying biophysics of light-triggered changes in bio-systems is crucial for cell viability and optimizing clinical applications of photo-induced processes in biotechnology, optogenetics and photopharmacology. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), we provide a holistic picture of light-triggered changes in membrane morphology, mechanics and dynamics. We combine microscopy of giant vesicles as minimal cell models, Langmuir monolayers, and molecular dynamics simulations. We employ giant vesicle elelctrodeformation as a facile and accurate approach to quantify the magnitude, reversibility and kinetics of light-induced area expansion/shrinkage as a result of azo-PC photoisomerization and content. Area increase as high as ~25% and a 10-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization. These results are in excellent agreement with simulations data and monolayers. Simulations also show that trans-to-cis isomerization of azo-PC decreases the membrane leaflet coupling. We demonstrate that light can be used to finely manipulate the shape and mechanics of photolipid-doped minimal cell models and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.
Optogenetics in the conventional sense, i.e. the use of engineered proteins that gain their light sensitivity from naturally abundant chromophores, represents an exciting means to trigger and control biological activity by light. As an alternate approach, photopharmacology controls biological activity with the help of synthetic photoswitches. Here, we used an azobenzene-derived lipid analogue to optically activate the transmembrane mechanosensitive channel MscL which responds to changes in the lateral pressure of the lipid bilayer. In this work, MscL has been reconstituted in nanodiscs, which provide a native-like environment to the protein and a physical constraint to membrane expansion. We characterized this photomechanical system by FTIR spectroscopy and assigned the vibrational bands of the light-induced FTIR difference spectra of the trans and cis states of the azobenzene photolipid by DFT calculations. Differences in the amide I range indicated reversible conformational changes in MscL as a direct consequence of light switching. With the mediation of nanodiscs, we inserted the transmembrane protein in a free standing photoswitchable lipid bilayer, where electrophysiological recordings confirmed that the ion channel could be set to one of its sub-conducting states upon light illumination. In conclusion, a novel approach is presented to photoactivate and control cellular processes as complex and intricate as gravitropism and turgor sensing in plants, contractility of the heart, as well as sensing pain, hearing, and touch in animals.
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