Affinity Purification and Functional Characterization of Dynamin-Related Protein 1

Abstract

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

Keywords: Dynamin-related protein; Electron microscopy; GTPase; Mitochondrial fission; Protein oligomerization.

Fig. 1

Purification of Drp1. (a) Affinity purification followed by size-exclusion chromatography (SEC) is used to isolate purified protein. (b) A representative gel highlights the yield and purity of Drp1 following affinity purification. (c and d) Following SEC, void and peak fractions are observed, and most of the protein resides within the peak

Fig. 2

Affinity tags impact Drp1 activity. (a) The GTPase activity of Drp1 with different affinity tags was measured using a colorometric assay that measures phosphate generation. (b) The calculated activity rates (k_obs) highlight the impact of distinct tags (CBP and GST) on Drp1 function

Fig. 3

Sedimentation assay can be used to quantify Drp1 oligomerization. (a) The sedimentation assay quantifies Drp1 self-assembly in the absence (−PCP) and presence (+PCP) of a non-hydrolyzable GTP analogue, GMPPCP. After centrifuging the sample, the supernatant and pellet fractions (left and right lanes, respectively) are run on a gel. (b) Imaging software was used to quantify the relative amount of Drp1 in the pellet

Fig. 4

Negative-stain election microscopy (EM) provides a qualitative assessment of Drp1 oligomerization. In the absence of nucleotide (top panels), Drp1 forms smaller multimers that appear as crescent-shaped particles. In the presence of GMPPCP (bottom panels), tagless Drp1 self-assembles into larger spiral structures. Both affinity tags (CBP and GST) limit the formation of functional assemblies. Scale bar, 100 nm