abs1 fragments  (New England Biolabs)

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Domain organization of Tmod1 and Lmods, and design of the Tmod1–Lmod2 c hybrid construct. Numbers under the diagrams indicate the boundaries of domains. For Tmod1, the helix of ABS1 (aa 67–81) and the LRR portion of ABS2 (aa 181–337) are highlighted. ( b ) Nucleation activity of full-length Lmod1, Lmod2 and the hybrid construct Tmod1–Lmod2 C as compared with Tmod1 and the Arp2/3 complex (25 nM, activated by 100 nM N-WASP WCA). The left two graphs show time courses of polymerization of 2 μM Mg–ATP–actin (6% pyrene labelled) in the presence of 25 nM of the indicated proteins (colour coded) or the buffer control (black). The graph on the right shows the concentration dependence of the polymerization rates, displayed as the mean of three experiments±s.e.m. ( c , d ) Contribution of the various domains of Lmod1 ( c ) and Lmod2 ( d ) to the nucleation activity. The graphs on the left and the right show, respectively, the time course of polymerization of 2 μM Mg–ATP–actin in the presence of 25 nM Lmod fragments (colour coded) or buffer (black) and the concentration dependence of polymerization rates.

Article Snippet: The ABS1 fragments were cloned between the SapI and XhoI sites of vector pTYB11 (NEB).

Techniques: Construct, Activity Assay, Concentration Assay

Journal: Nature Communications

Article Title: How Leiomodin and Tropomodulin use a common fold for different actin assembly functions

doi: 10.1038/ncomms9314

Figure Lengend Snippet: ( a ) Sequence conservation analysis of Tmod and Lmod (see also ). Fifty Tmod (purple) and fifty Lmod (orange) sequences were aligned separately or together (blue), and residue conservation scores were calculated with the program Scorecons and plotted on the human Tmod1 sequence (the scores of residues absent in Tmod1 are not shown). The Tmod1 diagram on top indicates the boundaries of the TM- and actin-binding sites. Diagrams on the bottom illustrate ABS1 constructs, and hybrid Tmod1 (magenta)/Lmod (green) ABS2 constructs (TL1 ABS2 , TL2 ABS2 and Tmod1 ABS2 Mut). The 11 residues of Tmod1 ABS2 replaced by their Lmod1 counterparts (highlighted in green across the conservation plots) tend to be conserved among Lmods, but poorly conserved between Lmods and Tmods. ( b ) Concentration dependence of polymerization rates of Lmod1 and Lmod2 in the absence (solid lines) or the presence (broken lines) of 1 μM TM, displayed as the mean of three experiments±s.e.m. ( c ) ITC titrations of ABS1 constructs (as indicated) into LatB-actin. The experimental conditions are listed for each experiment, including temperature and the concentrations of ABS1 constructs in the syringe and LatB-actin in the cell. Open symbols correspond to titrations into buffer. Only the titration of Tmod1 ABS1 could be fitted to a binding isotherm (red curve, fitting parameters inside graph), whereas Lmod1 ABS1 and Lmod2 ABS1 did not appear to bind (solid black symbols). Errors correspond to the s.d. of the fits.

Article Snippet: The ABS1 fragments were cloned between the SapI and XhoI sites of vector pTYB11 (NEB).

Techniques: Sequencing, Binding Assay, Construct, Concentration Assay, Titration