Protein folding and aggregation are in direct competition in living systems, yet measuring the two pathways simultaneously has rarely been accomplished. In order to identify the mechanism of high-pressure dissociation of aggregates, we compared the simultaneous on- and off-pathway. behavior following dilution of freshly denatured P22 tailspike protein. Tailspike assembly at 100 mug/mL was monitored at four temperatures using a combination of size-exclusion chromatography and native polyacrylamide gel electrophoresis (PAGE) and folding and aggregation rates and yields were determined. As temperature increased, the yield of native trimeric tailspike decreased from 26.1 +/- 1.3 mug/mL at 20degreesC to O mug/mL at 37degreesC. Pressure treatment dissociated 60% of the trapped aggregates created at 37degreesC and yielded 19.8 +/- 1.1 mug/mL of native trimer following depressurization and incubation at 20degreesC. The rate of refolding of "freshly denatured" tailspike was compared to that following pressure treatment. The trimer formation rate increased by a factor of roughly five, and the aggregate rate decreased by a factor of three, following pressure treatment. Circular dichroism and high-pressure intrinsic tryptophan fluorescence measurements support the model that a structured intermediate is formed in a rapid manner under high pressure from. a pressure-sensitive aggregate population. (C) 2003 Wiley Periodicals, Inc.