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. 2019 Jun 24;8:e44698. doi: 10.7554/eLife.44698

Figure 3. Global misfolding and oligomerization kinetics of Trp-less moPrP doped with 2 mol% of the unlabelled and TNB-labelled single Trp, single Cys-containing mutant variants of moPrP, monitored by CD and size-exclusion chromatography, respectively.

(A) None of the dopant proteins altered the global misfolding kinetics of Trp-less moPrP to a significant extent at the dopant concentrations used in these experiments. The total protein concentration was kept fixed at 100 µM in all experiments. Global misfolding was monitored using far-UV CD. Error bars are standard deviation of the mean, determined from three independent measurements, from three separate samples. The global misfolding rate, as determined from a single exponential fit of all the data was 0.09 ± 0.03 h−1. (B) Size-exclusion chromatograms at different time points of oligomerization of 100 µM Trp-less moPrP, showing the presence of oligomers OL, OS and monomer M. (C) Normalized monomer loss kinetics, estimated from (B). The black line through the data is shown as a guide to the eye.

Figure 3—source data 1. Raw data for Figure 3.
elife-44698-fig3-data1.xlsx (21.1KB, xlsx)
DOI: 10.7554/eLife.44698.009

Figure 3.

Figure 3—figure supplement 1. Trp-less moPrP shows a negligible change in fluorescence intensity upon oligomerization.

Figure 3—figure supplement 1.

Oligomerization of 98 µM of Trp-less moPrP shows no change in fluorescence signal over a period of 12 hr. This indicated that the contribution of scatter to the fluorescence signal at this wavelength (340 nm) was negligible. The average fluorescence signal was subtracted from all experiments to account for the background signal of 98 µM Trp-less moPrP, present in all samples, for suppression of inter-molecular FRET. Similar background correction was also made for samples containing dopant at a 1:99 doping ratio. Error bars are standard deviation of the mean, determined from three independent measurements.