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Membrane Curvature Revisited-the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy.

ABSTRACT: G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.

SUBMITTER: Fried SDE

PROVIDER: S-EPMC7896002 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 °C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.

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Membrane Curvature Revisited-the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy.

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