perimeter. The nano-scale clustering in turn is orchestrated by engaging with the underlying actin cytoskeletal machinery. The cytoskeletal interaction mediated by specific sites on the intra-cellular domain influences the organization and dynamics of the protein both at the nano-scale and meso-scale providing a selective engagement with actin filaments nucleated by formin-based actin nucleation machinery over Arp2/3. Additionally, the extra-cellular domain and its interaction with elements of the extra-cellular matrix also influence the dynamics of the protein and its nano as well as meso-scale organization at the plasma membrane. Taken together, our data captures a hierarchical nature of organization of CD44 at the cell surface with cytoskeleton-templated nano-scale clusters populating the meso-scale domains.
Rhodopsin is the G‐protein‐coupled receptor (GPCR) responsible for scotopic vision in the retina. Up to this point, the role of water in the activation of GPCRs has remained largely unknown. Recently, however, nanosecond molecular dynamics simulations have revealed an influx of bulk water into rhodopsin during activation [1]. Utilizing rhodopsin as a model GPCR, we tested the hypothesis that rhodopsin activation is hydration mediated using osmotic stress techniques. We subjected rhodopsin within its native lipid membranes to varying osmotic pressures induced by different‐sized polyethylene glycol polymers. UV‐Visible spectroscopy of the photoactivated rhodopsin system reveals the fraction of protein in the active metarhodopsin‐II (MII) conformation, the receptor state capable of activating the G‐protein. We discovered high‐molecular weight osmolytes uniformly favored the closed, inactive metarhodopsin‐I conformation by dehydration of the protein interior. By contrast, small osmolytes penetrated into the transducin binding cleft and stabilized the active MII conformation until a quantifiable saturation point. A universal osmotic response occurred in the limit of increasing osmolyte size and maximal polymer exclusion from rhodopsin. By measuring the thermodynamic dependence of the metarhodopsin equilibrium on osmotic pressure, we determined that rhodopsin activation is coupled to a bulk influx of 80–100 water molecules into the protein core with a substantial increase in compressibility. We propose a new model for the functional role of water in GPCR signal transduction, in which a wet‐dry cycling mechanism amplifies the activation of G‐proteins. Our results necessitate a new understanding of GPCR activation, in which the influx of water plays a critical role in establishing the active receptor conformation.Support or Funding InformationThis work was supported by the NIH (EY026041 and EY012049) and the NSF (MCB 1817862).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rhodopsin, a prototypical G-protein coupled receptor (GPCR), functions as a sensitive photon detector in retinal rod cells. Signal transduction by rhodopsin is achieved through the binding and release of the transducin G-protein. Despite the numerous recent structures of complexes with effector proteins, the mechanism underlying G-protein release from an activated GPCR remains elusive [1]. Here we propose that rhodopsin hydration/dehydration cycling facilitates transducin binding and release. The dissociation constant (K d ) between photoactivated rhodopsin and the high-affinity C-terminal analog of transducin (G alpha CT) was measured in response to osmotic stress. The active metarhodopsin-II (MII) fraction of rhodopsin was obtained using UV-visible spectrophotometry, and osmotic stress was generated using various molecular weight polyethylene glycol (PEG) polymers. Preliminary data indicate that large molecular weight polymers, such as PEG1500, are unable to enter the protein interior, and dehydrate the rhodopsin. The opposite effect is observed for small molecular weight polymers, which enter the protein core and have a hydrating effect. Large molecular weight polymers reduce the binding affinity for G alpha CT to rhodopsin, while small molecular weight polymers increase this binding affinity. Our results indicate that rhodopsin hydration is necessary for transducin binding. We therefore propose the sponge model for GPCR signaling, in which an influx of water during rhodopsin activation is necessary to stabilize the active MII conformation and to facilitate transducin binding. Formation of the high-affinity rhodopsintranducin complex displaces water from the protein interior, destabilizing the receptor. Nucleotide exchange further dehydrates the receptor, causing it to return to the inactive MI conformation, thus releasing transducin in the process. This wet/dry cycle can repeat many times for one photoexcitation event, explaining how many molecules of transducin can be activated by one molecule of rhodopsin. [1] U. Chawla, et al. (2016) Angew. Chem. Int. Ed. 55, 588-592.
Methyl-TROSY NMR of specifically 13 C methyl-labeled proteins associated to nanodisc bilayers is a powerful tool to probe the structure of membrane-bound macromolecular complexes. However, protein methyl resonances are obscured by baseline artefacts stemming from the strong signals of the lipid chains. While artefacts can be eliminated by using lipids with perdeuterated acyl chains, this strategy is prohibited when complex lipid mixtures are required for protein function. Here, we compared three approaches to selectively remove the lipid signals, using the ArfGAP ASAP1 PH domain and the small GTPase Arf1 as representative proteins. (I) Spin Label Induced PRE (SLIP). The bilayer was doped with a small fraction of doxyl spin labeled lipids at position 5, 10 or 16 along the acyl chain, or 25-doxyl cholesterol (CNO) to induce paramagnetic line broadening of the lipid resonances. (II) Selective excipient reduction and removal (SIERRA), a 1 H/ 13 C frequency selective filter proposed by Arbogast et al. (III) Lipid-Proton Driven Spin Diffusion (L-PDSD) from the lipid methylene signal to the lipid methyl signal. Addition of 2 mol% of 10-doxyl lipid or CNO attenuated both the lipid methylene and methyl signals by a factor of 5. In combination with L-PDSD, lipids signals were completely removed. Protein methyls were attenuated in a distance dependent fashion with severe broadening for methyl located at the membrane phosphate plane or below. On the contrary, application of a SIERRA filter caused only minor reduction of protein methyl resonances at high field, while attenuating the lipid methyl signal by a factor of 10. The improvement in data quality allowed us to determine the interface between Arf1 and ASAP1 at the membrane surface using solvent PRE and chemical shift perturbations.
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