We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 μM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 μM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.
Anionic phospholipids are essential structural components of cell membranes. Spatiotemporal dynamics of these lipids play central roles in regulating signalling events, membrane trafficking, maintenance of cell‐shape, and cargo transport. On the other hand, defects in anionic phospholipid metabolism are linked to multiple diseases. Hence, the ability to visualize these phospholipids and their dynamics in living cells can afford mechanistic insights into vital cell processes, guide the development of therapeutics, and lead to diagnostic agents. In this exciting backdrop, fluorescent sensors that can detect anionic phospholipids become key chemical tools that can be used to image and track these bio‐molecules in a confocal microscopy platform. In this review, we highlight existing chemical probes and sensing strategies for anionic phospholipids along with their pros and cons in the context of their applicability toward imaging and tracking these essential lipids in living cells.
Anionic phospholipids are key cell signal mediators. The distribution of these lipids on the cell membrane and intracellular organelle membranes guides the recruitment of signaling proteins leading to the regulation of cellular processes. Hence, fluorescent sensors that can detect anionic phospholipids within living cells can provide a handle into revealing molecular mechanisms underlying lipid-mediated signal regulation. A major challenge in the detection of anionic phospholipids is related to the presence of these phospholipids mostly in the inner leaflet of the plasma membrane and in the membranes of intracellular organelles. Hence, cell-permeable sensors would provide an advantage by enabling the rapid detection and tracking of intracellular pools of anionic phospholipids. We have developed a peptide-based, cell-permeable, water-soluble, and ratiometric fluorescent sensor that entered cells within 15 min of incubation via the endosomal machinery and showed punctate labeling in the cytoplasm. The probe could also be introduced into living cells via lipofection, which allows bypassing of endosomal uptake, to image anionic phospholipids in the cell membrane. We validated the ability of the sensor toward detection of intracellular anionic phospholipids by colocalization studies with a fluorescently tagged lipid and a protein-based anionic phospholipid sensor. Further, the sensor could image the externalization of anionic phospholipids during programmed cell death, indicating the ability of the probe toward detection of both intra- and extracellular anionic phospholipids based on the biological context.
opened a general discussion of the paper by Amitabha Chattopadhyay: The analysis depends on the spatial resolution, so when you classify them as dimers or trimers they have to be within a certain volume, so can you say that they are really dimers or are they just very close to each other? Amitabha Chattopadhyay responded: The analysis depends on spatial correlation of the photobleaching data. The size of the oligomers is deduced from tting the model. Amit Sharma asked: What is the size of these clusters when they are dimers or trimers? Would it not be possible to obtain the size through FRET studies? Amitabha Chattopadhyay answered: The size of the oligomers will be in the nm range. FRET will provide proximity (distance) information, not size. Rajaram Swaminathan said: I have two questions. First, what role does lipid composition play in the results shown? Will results be different with another lipid composition? Second, are there reports in the literature on GPCR oligomerization in articial liposomes where composition can be controlled? Amitabha Chattopadhyay responded: To answer the rst question, results from our laboratory and a number of groups have shown that lipid composition could play a role in GPCR function and oligomerization. To the second question, there are not too many examples of GPCRs oligomers studied in reconstituted liposomes. This is due to a variety of reasons which include difficulty in purication, and more importantly, lack of other cellular ingredients (necessary for oligomerization) in liposomal systems.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.