PMID: 20923645;PMCID: PMC3042581;Abstract:
Alpha-synuclein (aS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αSis abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length aS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the aS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu. © 2010 by the Biophysical Society.
PMID: 15803674;Abstract:
The influences of lanosterol on lipid bilayers have been compared to those of cholesterol by combining deuterium ( 2H) NMR spin relaxation studies with segmental order parameter measurements. For bilayers of 1,2-diperdeuteriomyristoyl-sn-glycero-3-phosphocholine (DMPC-d 54), the results are consistent with a square-law dependence of the 2H Zeeman relaxation rates (R 1Z) on the corresponding order parameters (S CD). This behavior is indicative of relatively slow order fluctuations, for example, due to quasi-elastic bilayer disturbances. Significant differences are found in the influences of lanosterol versus cholesterol on the microscopic NMR observables; although lanosterol produces smaller order parameters than cholesterol, it leads to larger relaxation rates. By correlating the NMR relaxation behavior with the order parameters, the results are explained by a progressive reduction of the bilayer elasticity, which parallels the biosynthetic pathway from lanosterol to cholesterol.
PMID: 2025300;Abstract:
Bovine rhodopsin was recombined with various phospholipids in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the headgroups was varied. The ratio of MII / MI produced in the recombinant membrane vesicles by an actinic flash was studied as a function of pH, and compared to the photochemical activity observed for rhodopsin in native ROS membranes. MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond timescale. Recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but demonstrated titration curves with different pK values. The presence of phosphoethanolamine or phosphoserine headgroups increased the amount of MII produced. In the case of phosphatidylserine this may result from alteration of the membrane surface potential, leading to an increase in the local H+ activity. The results indicate that the Gibbs free energies of the MI and MII conformational states are influenced by the membrane bilayer environment, suggesting a possible role of lipids in visual excitation. © 1991 Academic Press, Inc.
PMID: 23701524;Abstract:
We have explored the relationship between conformational energetics and the protonation state of the Schiff base in retinal, the covalently bound ligand responsible for activating the G protein-coupled receptor rhodopsin, using quantum chemical calculations. Guided by experimental structural determinations and large-scale molecular simulations on this system, we examined rotation about each bond in the retinal polyene chain, for both the protonated and deprotonated states that represent the dark and photoactivated states, respectively. Particular attention was paid to the torsional degrees of freedom that determine the shape of the molecule, and hence its interactions with the protein binding pocket. While most torsional degrees of freedom in retinal are characterized by large energetic barriers that minimize structural fluctuations under physiological temperatures, the C6-C7 dihedral defining the relative orientation of the β-ionone ring to the polyene chain has both modest barrier heights and a torsional energy surface that changes dramatically with protonation of the Schiff base. This surprising coupling between conformational degrees of freedom and protonation state is further quantified by calculations of the pKa as a function of the C6-C7 dihedral angle. Notably, pKa shifts of greater than two units arise from torsional fluctuations observed in molecular dynamics simulations of the full ligand-protein-membrane system. It follows that fluctuations in the protonation state of the Schiff base occur prior to forming the activated MII state. These new results shed light on important mechanistic aspects of retinal conformational changes that are involved in the activation of rhodopsin in the visual process. © 2013 American Chemical Society.
