In dopaminergic neurons, a-synuclein (αS) partitions between a disordered cytosolic state and a lipidbound state. Binding of αS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free and lipid-bound αS state, and between the different bound states, is slow on the NMR timescale, being in the range of 1-10 s −1 . Partitioning of the binding modes is dependent on the lipid: αS stoichiometry, and tight binding with slow exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the Nterminus of αS adopts an α-helical conformation while succeeding residues retain characteristics of a random coil. The C-terminal 40 residues remain dynamically disordered, even at high lipid concentration, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamical properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide NOE buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr 39 indicates an ordered environment for this "dark state." Titration of αS with increasing amounts of lipids suggests that the binding mode under high lipid conditions remains qualitatively similar to the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface, and instead suggest that considerable remodeling of the vesicles is induced by αS.

Three familial variants of the presynaptic protein α-synuclein (αS), A30P, E46K, and A53T, correlate with rare inherited Parkinson’s disease (PD), while wild-type αS is implicated in sporadic PD. The classic manifestation of both familiar and sporadic PD is the formation of fibrillar structures of αS which accumulate as the main component in intraneuronal Lewy bodies. At presynaptic termini, the partitioning of αS between disordered cytosolic and membrane-bound states likely mediates its proposed role in regulation of reserve pools of synaptic vesicles. Previously, we reported on multiple distinct phospholipid binding modes of αS with slow binding kinetics. Here, we report the phospholipid binding properties of the disease variants, viewed by solution NMR in a residue-specific manner. Our results agree qualitatively with previous biophysical studies citing overall decreased lipid affinity for the A30P mutation, comparable affinity for A53T, and an increased level of binding of E46K, relative to wild-type αS. Additionally, our NMR results describe the distribution of lipid-bound states for αS: the population of the SL1 binding mode (residues 3−25 bound as a helix) is augmented by each of the disease variants, relative to wild-type αS. We propose that the SL1 binding mode, which anchors the N-terminus of αS in the lipoprotein complex while the hydrophobic NAC region remains dynamically disordered, is prone to intermolecular interactions which progress toward disease-associated oligomers and fibrils. The elevation of the SL1 binding mode, unchecked by a proportionate population of binding modes incorporating the full N-terminal domain, may well account for the increased toxicity of the A30P, E46K, and A53T disease variants of αS.

Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left-right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20 -25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46 -60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis. Developmental Dynamics 235:3438 -3447, 2006.

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Polyphenylalkene derivatives Q 0740Suzuki and Heck Coupling Reactions Mediated by Palladium Complexes Bearing trans-Spanning Diphosphines. -Ten aryl halides of varying electron density are examined in Heck reactions with styrene and Suzuki coupling reactions with phenylboronic acid using terphenyl-based diphosphine palladium complexes (I) as catalysts.In Heck reactions, complexes (Ia) and (Ib) are found to be effective catalysts, whereas complex (Ic) is proved to be ineffective. Only (E)-alkenes are formed in the presence of complex (Ib), whereas (E)/(Z)-mixtures are obtained when complex (Ia) is employed. All complexes (I) are found to be effective catalysts in Suzuki coupling reactions. -(SMITH, R. C.; BODNER, C. R.; EARL, M. J.; SEARS, N. C.; HILL, N. E.; BISHOP, L. M.; SIZEMORE, N.; HEHEMANN, D. T.; BOHN, J. J.; PROTASIEWICZ*, J. D.; J. Organomet. Chem. 690 (2005) 2, 477-481; Dep. Chem., Case West. Reserve Univ., Cleveland, OH 44106, USA; Eng.) -Klein

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