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The structure and dynamics of two partially folded states of apomyoglobin have been characterized at equilibrium using multi-dimensional NMR spectroscopy. Residue-specific measurements of chemical shift and internal dynamics in these states and in the native apoprotein and holoprotein indicate progressive accumulation of secondary structure and increasing restriction of backbone dynamics as the chain collapses to form increasingly compact states. Under weakly folding conditions, the polypeptide fluctuates between unfolded states and local elements of structure that become extended and stabilized as the chain becomes more compact. These results provide a detailed model for molecular events that are likely to occur during folding of myoglobin.

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Background:The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native ␣-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric ␣-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease.

␣-Synuclein (␣-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related ␣-synulceinopathies. However, whether phosphorylation promotes or inhibits ␣-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of ␣-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type ␣-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated ␣-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of ␣-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of ␣-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern ␣-syn aggregation and toxicity in PD and related ␣-synulceinopathies.

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Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of ␣-synuclein (␣-syn) in Parkinson disease. Herein we demonstrate that ␣-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated ␣-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P ␣-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membranebinding properties of monomeric ␣-syn. These studies demonstrated that phosphorylation at S87 expands the structure of ␣-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of ␣-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in ␣-syn normal biology.