Skip to main content
. 2022 Aug 1;5(12):e202101284. doi: 10.26508/lsa.202101284

Figure 6. Patient DRP1 variants alter DRP1 assembly-state and melting temperature.

Figure 6.

(A) SEC-MALS analysis of WT DRP1 (purple trace), DRP1 G363D (green trace), DRP1 G401S (orange trace), and DRP1 R710G (magenta trace) to assess for differences in multimeric distributions. Overlay of normalized differential refractive index of all protein samples (200 μg, 2.0 mg/ml) with peaks corresponding to monomeric, dimeric, and tetrameric oligomer species labelled as determined by predicted molecular masses of each multimeric species. Data normalized and scaled to allow for easier comparison because of slight differences in amount of protein loaded onto the column. (B) Melt curves of WT DRP1 and patient variants. Thermafluor analysis of protein unfolding of WT DRP1 (5.0 μM) and three patient variants (G363D, G401S, and R710G) either alone (black, dotted line), in the presence of 500 μM GDP (dark grey, dashed line), or GMP-PNP (light grey, solid line). Data plotted as the first derivative of the fluorescence signal with respect to time. (C, D) Tm values determined from the temperature corresponding to the maximum fluorescence value in the absence of and presence of 500 μM GDP or GMP-PNP. (C) Thermafluor analysis of the first protein unfolding event reported as the melting temperature (Tm) of WT DRP1 (5.0 μM) and three patient variants either alone, or in the presence of 500 μM GDP or GMP-PNP. (D) Thermafluor analysis of the second protein unfolding event reported as the melting temperature (Tm). Only WT and R710G shown as they are the only two constructs with a prominent second unfolding event. Data are representative of two independent experiments, each with three technical replicates. ***P < 0.00001. Differences between Tm values of all constructs alone in comparison with constructs with 500 μM GDP or 500 μM GMP-PNP significant to P < 0.003. Tm values of all constructs with 500 μM GDP in comparison to 500 μM GMP-PNP are significant to P < 0.03 except for R710G with 500 μM GDP in comparison to R710G with 500 μM GMP-PNP where P < 0.0003.