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. 2011 Dec 1;7(12):e1002396. doi: 10.1371/journal.pgen.1002396

Figure 6. Oligomerization of plectin's RD.

Figure 6

(A) Schematic representation of plectin's domain structure with exon allocation of subdomains and recombinant proteins expressed in baculovirus. 1's stands for 11 first exons alternatively spliced into exon 2. Highlighted are plectin's actin-binding domain (orange), the 9 spectrin repeats (green), the α-helical rod domain (light blue), and the C-terminal domain comprising 6 plectin repeats (dark blue). Red star, position of the Ogna mutation. pNV10 and pNV11, recombinant His-tagged wild-type and Ogna plectin RDs (135 kDa); pNV14 and pNV15, corresponding GST-tagged versions (158 kDa); pHLH20/wt and pHLH20/Ogna, recombinant His-tagged wild-type and Ogna versions of the RD flanked by the 9th spectrin repeat and the linker region between plectin's rod and the C-terminal region (170 kDa). (B) BN-(4–10%)-PAGE of recombinant wild-type (WT) and Ogna (O)-RDs using ferritin (440 and 880 kDa) as size marker. (C) Chemical cross-linking of RDs. Purified RD samples before and after cross-linking with DMS were analyzed by SDS-5%-PAGE. Note that i) the major cross-linked species (upper arrow) migrated just above the ∼500 kDa size marker (plectin), and ii) a fraction of both proteins could not be cross-linked in solution and was detected as monomers (lower arrow). (D) Molecular mass measurement by SEC-MALS analysis. The blue line traces the absorbance at 280 nm of the eluate from a Superose 6 10/300GL column as a function of time. The red dotted line represents the weight-average molecular weight of the species in the eluate, calculated from refractive index and light-scattering measurements. The protein peaks containing RD dimers and high molecular mass polymers are labeled with d and p, respectively. (E,F) Dissociation of plectin RD oligomers as a function of temperature and urea concentration. Samples were preincubated at increasing temperatures or concentrations of urea, cross-linked with DMS, and resolved by SDS-PAGE. The relative percentages of oligomers in each sample, determined by densitometric analysis of the gel lanes, were plotted as a function of temperature (E) or added urea (F). Data are shown as mean ±SD of three independent experiments performed in duplicates. The solid lines show the linear regression fit of the data (r2≥0.9689).