Optically sensing phospholipid induced coil–helix transitions in the phosphoinositide-binding motif of gelsolin

Abstract

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.

Regulation of actin assembly by PI(4,5)P2 and other inositol phospholipids: An update on possible mechanisms

“…Effects of PI(3,4,5)P3 on sorting nexin 9 can also promote its stimulation of actin assembly by activation of N-Wasp/Arp2.3 [52–54]. The requirement for high curvature suggests that the conformation of PI(4,5)P2 or another phosphoinositide, which generally have larger headgroups than other bilayer lipids [55, 56], might be more accessible to the proteins they regulate, as seen in the large effect of submicron membrane curvature on the enzymatic acidity of PI3-kinase [57]. Alternatively high curvature might alter the ionization state of PI(4,5)P2, as predicted from the effects of lateral packing density on the net charge of PI(4,5)P2 and other inositol lipids [58].…”

A Peptide-Based Fluorescent Sensor for Anionic Phospholipids

“… 3 Electrostatic interactions of negatively charged polar headgroups of anionic phospholipids with positively charged interfaces of cytoplasmic proteins form the basis of lipid–protein interactions at the membrane cytosol interface. 12 , 13 These initial interactions lead to downstream signaling events that regulate cellular processes. 9 …”

Fluorescent Chemical Tools for Tracking Anionic Phospholipids

“…As passive regulators, binding of an effector protein to a cluster of closely spaced phospholipids can increase the local concentration of the protein allowing other signalling proteins to bind to the effector [1d,3,4d] . As active regulators, phospholipid binding can cause a conformational change in a protein leading to its activation [1b,4d,5e,8] . Membrane binding of signalling proteins is governed by both specific interactions with lipid headgroups and non‐specific interactions with the membrane.…”

“…Membrane binding of signalling proteins is governed by both specific interactions with lipid headgroups and non‐specific interactions with the membrane. Peripheral proteins which bind to anionic phospholipids generally contain domains with either positively charged poly‐basic amino acids or cationic metal ion centers [5e,8–9] . These domains recognize the negatively charged membrane surface via long‐range electrostatic attractive forces [1b,10] .…”

“…In the past two decades, there have been significant advances in the development of fluorescent tags that have allowed visualization of various molecular processes within optically transparent living systems [28a,30] . Fluorescent sensors and probes for sensing phospholipids have featured as key chemical tools for deciphering fundamental mechanisms underlying phospholipid mediated cellular regulation [4c,5e,6e,f,31] . In this review, we highlight emerging fluorescent chemical tools for tracking and imaging anionic phospholipids in the backdrop of open questions as well as current limitations in phospholipid detection.…”