␣-Synuclein is a highly conserved presynaptic protein of unknown function. A mutation in the protein has been causally linked to Parkinson's disease in humans, and the normal protein is an abundant component of the intraneuronal inclusions (Lewy bodies) characteristic of the disease. ␣-Synuclein is also the precursor to an intrinsic component of extracellular plaques in Alzheimer's disease. The ␣-synuclein sequence is largely composed of degenerate 11-residue repeats reminiscent of the amphipathic ␣-helical domains of the exchangeable apolipoproteins. We hypothesized that ␣-synuclein should associate with phospholipid bilayers and that this lipid association should stabilize an ␣-helical secondary structure in the protein. We report that ␣-synuclein binds to small unilamellar phospholipid vesicles containing acidic phospholipids, but not to vesicles with a net neutral charge. We further show that the protein associates preferentially with vesicles of smaller diameter (20 -25 nm) as opposed to larger (ϳ125 nm) vesicles. Lipid binding is accompanied by an increase in ␣-helicity from 3% to approximately 80%. These observations are consistent with a role in vesicle function at the presynaptic terminal.

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The reversible cold, heat, and pressure unfolding of RNase A and RNase A--inhibitor complex were studied by 1D and 2D 1H NMR spectroscopy. The reversible pressure denaturation experiments in the pressure range from 1 bar to 5 kbar were carried out at pH 2.0 and 10 degrees C. The cold denaturation was carried out at 3 kbar, where the protein solution can be cooled down to -25 degrees C without freezing. Including heat denaturation experiments, the experimental data obtained allowed us to construct the pressure--temperature phase diagram of RNase A. The experimental results suggest the possibility that all three denaturation processes (cold, heat, and pressure) lead to non-cooperative unfolding. The appearance of a new histidine resonance in the cold-denatured and pressure-denatured RNase A spectra, compared to the absence of this resonance in the heat-denatured state, indicates that the pressure-denatured and cold-denatured states may contain partially folded structures that are similar to that of the early folding intermediate found in the temperature-jump experiment reported by Blum et al. [Blum, A. D., et al. (1978) J. Mol. Biol. 118, 305]. A hydrogen-exchange experiment was performed to confirm the presence of partially folded structures in the pressure-denatured state. Stable hydrogen-bonded structures protecting the backbone amide hydrogens from solvent exchange were observed in the pressure-denatured state. These experimental results suggest that the pressure-denatured RNase A displays the characteristics of a the inhibitor 3'-UMP show that the RNase A-inhibitor complex is more stable than RNase without the inhibitor.