Kinetic studies of protein L aggregation and disaggregation.
We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.
Cellmer
Troy
T
Department of Chemical Engineering, University of California, Berkeley, Berkeley, CA 94720, United States.
Douma
Rutger
R
Huebner
Ansgar
A
Prausnitz
John
J
Blanch
Harvey
H
D000686:Amyloidosis; D001426:Bacterial Proteins / Q000378:metabolism*; D052160:Benzothiazoles; D003224:Congo Red; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012996:Solutions; D013844:Thiazoles
D001426:Bacterial Proteins; D052160:Benzothiazoles; D012996:Solutions; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red