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. 2020 Sep 8;10:14777. doi: 10.1038/s41598-020-71655-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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SEC-MALS and EM reveal altered quaternary structure and higher-order oligomerization states for mEGFP-Drp1 in vitro. (A,B) SEC-MALS profiles of Drp1-wt (A) and mEGFP-Drp1 (B) as a function of total loaded protein concentration before sieving. The horizontal dashed line in each plot indicates the approximate molecular mass of a corresponding tetramer (~ 328 kDa for Drp1-wt and ~ 444 kDa for mEGFP-Drp1). For Drp1-wt, molar mass distribution for only 8.5 μM loading protein concentration is shown. Drp1-wt displayed elution profile shifts as a function of protein concentration typically observed for self-associating systems in dynamic equilibrium. mEGFP-Drp1 showed no such apparent elution profile shifts and primarily sampled tetramer or higher oligomeric states. (C,D) Representative negative-stain EM images of Drp1-wt (C) and mEGFP-Drp1 (D) in the presence of GMP-PCP. Scale bar, 200 nm. Insets show magnified images of representative self-assembled higher-order structures. Inset scale bar, 100 nm. (E,F) Boxplots of helical diameter (E) and lengths (F) comparing Drp1-wt and mEGFP-Drp1 in the presence of GMP-PCP. Statistically significant differences between groups are indicated by a star. Mean ± S.D. is indicated next to each box. For helical diameters in panel E, Drp1-wt n = 114; mEGFP-Drp1 n = 105, p < 0.0001. For helical lengths in panel F, Drp1-wt n = 116; mEGFP-Drp1 n = 117, p < 0.0001. SEC-MALS profiles and box and whisker plots were prepared using matplotlib v3.2.2 available at https://matplotlib.org/3.2.2/api/_as_gen/matplotlib.pyplot.boxplot.html.