PMID: 23583776;Abstract:
Misfolding and aggregation of the intrinsically disordered protein α-Synuclein (αS) in Lewy body plaques are characteristic markers of late-stage Parkinson's disease. It is well established that membrane binding is initiated at the N-terminus of the protein and affects biasing of conformational ensembles of αS. However, little is understood about the effect of αS on the membrane lipid bilayer. One hypothesis is that intrinsically disordered αS alters the structural properties of the membrane, thereby stabilizing the bilayer against fusion. Here, we used two-dimensional 13C separated local-field NMR to study interaction of the wild-type α-Synuclein (wt-αS) or its N-terminal (1-25) amino acid sequence (N-αS) with a cholesterol-enriched ternary membrane system. This lipid bilayer mimics cellular raft-like domains in the brain that are proposed to be involved in neuronal membrane fusion. The two-dimensional dipolar-recoupling pulse sequence DROSS (dipolar recoupling on-axis with scaling and shape preservation) was implemented to measure isotropic 13C chemical shifts and 13C-1H residual dipolar couplings under magic-angle spinning. Site-specific changes in NMR chemical shifts and segmental order parameters indicate that both wt-αS and N-αS bind to the membrane interface and change lipid packing within raft-like membranes. Mean-torque modeling of 13C-1H NMR order parameters shows that αS induces a remarkable thinning of the bilayer (≈ 6 Å), accompanied by an increase in phospholipid cross-sectional area (≈ 10 Å2). This perturbation is characterized as membrane annealing and entails structural remodeling of the raft-like liquid-ordered phase. We propose this process is implicated in regulation of synaptic membrane fusion that may be altered by aggregation of αS in Parkinson's disease. © 2013 Elsevier Ltd.
PMID: 17951745;Abstract:
Molecular fluctuations are a dominant feature of biomembranes. Cellular functions might rely on these properties in ways yet to be determined. This expectation is suggested by the fact that membrane deformation and rigidity, which govern molecular fluctuations, have been implicated in a number of cellular functions. However, fluctuations are more challenging to measure than average structures, which partially explain the small number of dedicated studies. Here, it is shown that two accessible laboratory methods, small-angle X-ray scattering and solid-state deuterium nuclear magnetic resonance (NMR), can be used as complementary probes of structural fluctuations in lipid membranes. In the case of X-ray scattering, membrane undulations give rise to logarithmically varying positional correlations that generate scattering peaks with long (power-law) tails. In the case of 2H NMR spectroscopy, fluctuations in the magnetic-coupling energies resulting from molecular motions cause relaxation among the various spin energy levels, and yield a powerful probe of orientational fluctuations of the lipid molecules. A unified interpretation of the combined scattering and 2H NMR data is provided by a liquid-crystalline membrane deformation model. The importance of this approach is that it is possible to utilize a microscopic model for positional and orientational observables to calculate bulk material properties of liquid-crystalline systems. © Humana Press Inc.
PMID: 22280374;PMCID: PMC3299983;Abstract:
We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations - as well as their extensive fluctuations - suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin. © 2012 American Chemical Society.
