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S1Supplemental Data How Capping Protein Binds the Barbed End of the Actin Filament

By Martin A. Wear, Atsuko Yamashita, Kyoungtae Kim and John A. Cooper


sion at 386 nm. Actin was used at a final concentration of 2.0 M (5%–10 % pyrene labeled) in 1 polymerization buffer (10 mM Imid-Construction of Deletion and Substitution CP Mutants A tandem CP bacterial expression vector (pET-3d/CP), allowing azole [pH 7.0], 50 mM KCl, 1 mM MgCl2, 0.2 mM ATP, 0.5 mM DTT, 0.2 mM CaCl2, 1 mM EGTA). Ca2-G-actin was “primed ” for 90 sfor cotranslation of chicken 1 and 1 subunits from a single plas-mid, was a kind gift from Dr. Takashi Obinata [S1]. Plasmids for prior to the initiation of polymerization by adding a 1/10th volume of 10 mM EGTA, 1 mM MgCl2 to exchange Ca2 for Mg2. A 1/20thexpression of mutants were created by PCR using the wt plasmid as a template and the QuikChange Site-Directed Mutagenesis Kit volume of 20 polymerization buffer (200 mM Imidazole [pH 7.0], 1 M KCl, 20 mM MgCl2, 20 mM EGTA) was then added to initiate(Stratagene). The resulting constructs were verified by sequencing the entire coding region in both directions. Oligonucleotide primers polymerization. CP species, GST-fusion proteins, or the free pep-tides were in 10 mM Imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl2,are listed in the Supplemental Table S1. pET-3d/CP(C34), en-coding a 1 C-terminal 34 amino acid deletion mutant (codon R244 0.5 mM DTT, 1 mM EGTA. CP species were added to the actin mixture immediately after priming followed by addition of 20 poly-to a stop), was constructed using the forward primer 1 and the reverse primer 2. pET-3d/CP(C28), encoding a 1 C-terminal 28 merization buffer and finally by addition of SAS. The solution was mixed and placed in the fluorometer, with a total dead time ofamino acid deletion mutant (codon P250 to a stop), was constructed using the forward primer 3 and the reverse primer 4. pET-3d / 12–15 s

Year: 2015
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