It is of great interest to determine how solutes such as urea, sugars, guanidinium salts, and trimethylamine N-oxide affect the stability, solubility, and solvation of globular proteins. A key hypothesis in this field states that solutes affect protein stability indirectly by making or breaking water structure. We used a new technique, pressure perturbation calorimetry, to measure the temperature dependence of a solute's partial compressibility. Using fundamental thermodynamic relations, we converted these data to the pressure dependence of the partial heat capacity to examine the impact of protein stabilizing and denaturing solutes on water structure by applying the classic two-state mixture model for water. Contrary to widely held expectations, we found no correlation between a solute's impact on water structure and its effect on protein stability. Our results indicate that efforts to explain solute effects should focus on other hypotheses, including those based on preferential interaction and excluded volume.

The effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, ␣-synuclein and acid-denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of ␣-synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that ␣-synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid-denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that ␣-synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.

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Abstract␣-Synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of ␣-synuclein in vitro, the specific amino acid types of ␣-synuclein involved in this type of aggregation have not been identified. We show, using human cytochrome c plus H 2 O 2 as the source oxidative stress, that the tyrosines of ␣-synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.Keywords: aggregation; Lewy bodies; oxidative stress; Parkinson's disease; ␣-synuclein; tyrosine Parkinson's disease is one of the most common age-related neurodegenerative disorders, affecting almost 3% of the population more than 65 years of age. This devastating disease, clinically characterized by tremor, rigidity, and bradykinesia, affects more than one million persons in the United States alone (Lang and Lozano 1998). The symptoms are caused by the loss of dopaminergic neurons in the substantia nigra region of the brain. The few surviving neurons in this region contain Lewy bodies (Holdorff 2002), intracytoplasmic aggregates composed largely of the protein ␣-synuclein (Spillantini et al. 1997).␣-Synuclein became the focus of Parkinson's disease research after the discovery of two rare, familial forms of the disease caused by point mutations in the ␣-synuclein gene (Polymerpoulos et al. 1997). Although the exact functions of ␣-synuclein are unknown, two main biochemical activities have been proposed: regulation of dopamine neurotransmission and regulation of dopaminergic synaptic vesicles. ␣-Synuclein is natively disordered (Weinreb et al. 1996) and has a random-coil circular dichroism spectrum (Kim 1997), but its ␣-helical content increases from 3% to 63%-70% on binding lipid membranes (Davidson et al. 1998).The cause of ␣-synuclein aggregation to form Lewy bodies is unknown. However, the production of reactive oxygen species (ROS) has been suggested as a central event. Oxidative stress is an intracellular imbalance in the pro-oxidant/ antioxidant equilibrium, favoring pro-oxidants (Sies 1991). The main source of intracellular ROS is the mitochondria (Boveris et al. 1972;Boveris and Chance 1973), where ROS are produced during the reduction of oxygen to water. Under conditions of oxidative stress, both ROS and the respiratory chain protein cytochrome c can leak from the mitochondria into the cytoplasm (Shigenaga et al. 1994). It has been suggested that this leakage is facilitated by pores in the mitochondrial membranes formed by ␣-synuclein (Lashuel et al. 2002;Volles and Lansbury Jr. 2002). Whatever the mechanism, cytochrome c co-localizes with ␣-synuclein in Lewy bodies (Hashimoto et al. 1999).All cellular components are vulnerable to oxidative stress. The damage to proteins includes side chain modification and main chain fragmentation. Two biological markers for oxidative damage in pr...