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Pressure treatment of tailspike aggregates rapidly produces on‐pathway folding intermediates
Author(s) -
Lefebvre Brian G.,
Robinson Anne Skaja
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10607
Subject(s) - chemistry , trimer , circular dichroism , dissociation (chemistry) , biophysics , chromatography , protein aggregation , folding (dsp implementation) , fluorescence , tryptophan , protein folding , population , dimer , biochemistry , organic chemistry , amino acid , demography , sociology , physics , quantum mechanics , electrical engineering , biology , engineering
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 μg/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 μg/mL at 20°C to 0 μg/mL at 37°C. Pressure treatment dissociated 60% of the trapped aggregates created at 37°C and yielded 19.8 ± 1.1 μg/mL of native trimer following depressurization and incubation at 20°C. 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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 595–604, 2003.