Mechanism of Prion Propagation: Amyloid Growth Occurs by Monomer Addition
Figure 2
Kinetics of NM Fiber Growth Support a Monomer Addition Model
(A) Initial rate of polymerization versus concentration of NM. Soluble NM at the indicated final concentrations was mixed with sonicated fibers (2.5 μM fibers at 5% of final volume) and polymerization was followed by a continuous thioflavin T assay. Rates shown were determined by the initial slopes of polymerization curves.
(B) Initial rate of polymerization versus concentration of soluble NM in the presence of sonicated seed (1% of final volume) measured by a discrete thioflavin T binding assay. Error bars throughout represent the standard deviation of at least three measurements.
(C) Polymerization of NM labeled with Alexa-647 at a C-terminal cysteine was monitored by the quenching of Alexa-647 fluorescence. The indicated concentrations of soluble NM were mixed with sonicated fibers (4% of final volume), and the initial rate of polymerization was measured.
(D) Initial rate of polymerization versus concentration of seed. Soluble NM (2.5 μM final concentration) was mixed with the indicated quantity of sonicated seed. Highly fragmented fibers were used to maximize the absolute rate. Rates were measured as in (A).
(E) Initial rate of polymerization versus concentration of seed as measured by discrete thioflavin T binding assay. Initial soluble NM concentration was 2.5 μM.
(F) Initial rate of polymerization versus concentration of seed as measured by Alexa-647 fluorescence quenching. Initial soluble NM concentration was 200 nM.