tl440m  (Cytoskeleton Inc)

Journal: The EMBO Journal

Article Title: Elongator is a microtubule polymerase selective for polyglutamylated tubulin

doi: 10.1038/s44318-024-00358-0

Figure Lengend Snippet: Reagents and tools table

Article Snippet: AMCA-Porcine tubulin , Cytoskeleton , TL440M.

Techniques: Recombinant, Sequencing, Protease Inhibitor, Software

Journal: ACS central science

Article Title: Electronic Energy Migration in Microtubules.

doi: 10.1021/acscentsci.2c01114

Figure Lengend Snippet: Figure 1. The structure of microtubules from a lattice of tubulin. (A) The tubulin dimer with tryptophan residues marked in red; the C- termini “tails” can be seen protruding from each monomer. (B) The structure of a microtubule, showing constituent arrangement of tubulin dimers, and the presence of a “seam”. (C) The repeating “lattice” of tubulin dimers in a microtubule.

Article Snippet: AMCA labeled tubulin solution was prepared by reconstituting lyophilized AMCA labeled tubulin powder (TL440m; Cytoskeleton Inc., Denver, CO, USA) in BRB80 supplemented with 10% glycerol and 1 mM GTP, to a final concentration of 45 μM tubulin.

Techniques:

Journal: ACS central science

Article Title: Electronic Energy Migration in Microtubules.

doi: 10.1021/acscentsci.2c01114

Figure Lengend Snippet: Figure 2. Steady-state spectra of tubulin and microtubules. (A) Absorbance spectra of free tubulin (teal) and free AMCA (purple) in solution. Intensity normalized fluorescence spectra of (B), free DL tryptophan in solution showing highest fluorescence emission at excitation wavelengths between 270 and 300 nm. (C) Unpolymerized GTP-tubulin, (D) microtubules polymerized using GTP-tubulin, (E) free AMCA, and (F) microtubules polymerized using AMCA-labeled GTP-tubulin. An energy transfer peak not observed in Figure 1A,B (at excitation 280−300 nm, and emission at 420−450 nm) is clearly visible. Colors represent photoluminescence intensity.

Article Snippet: AMCA labeled tubulin solution was prepared by reconstituting lyophilized AMCA labeled tubulin powder (TL440m; Cytoskeleton Inc., Denver, CO, USA) in BRB80 supplemented with 10% glycerol and 1 mM GTP, to a final concentration of 45 μM tubulin.

Techniques: Fluorescence, Labeling

Journal: ACS central science

Article Title: Electronic Energy Migration in Microtubules.

doi: 10.1021/acscentsci.2c01114

Figure Lengend Snippet: Figure 3. Confirmation and polymerization of tubulin polymorphs. Negative stain electron microscopy of tubulin polymorphic geometries for (A) free GTP-tubulin in solution, (B) GTP-tubulin polymerized 13 protofilament microtubules, (C) free GTP-tubulin oligomers polymerized using 100 μM vinblastine in solution, and (D) GMPCPP-tubulin polymerized 14 protofilament microtubules. Insets show schematics of tubulin polymer structures. Methodology used to perform negative staining is described in SI Appendix. Due to MAPs and drugs that induce microtubule bundling being absent in our solutions and the highly charged C-termini tail of tubulin (expected to cause intermicrotubule repulsion), intermicrotubule separation distances were large enough in solution for electronic energy transfer to be insignificant. (E) Routine to polymerize microtubules with different AMCA labeled tubulin: unlabeled tubulin ratios, thus different AMCA concentrations. See SI Appendix for protocols used to assemble different tubulin polymerization states and perform electron microscopy.

Article Snippet: AMCA labeled tubulin solution was prepared by reconstituting lyophilized AMCA labeled tubulin powder (TL440m; Cytoskeleton Inc., Denver, CO, USA) in BRB80 supplemented with 10% glycerol and 1 mM GTP, to a final concentration of 45 μM tubulin.

Techniques: Staining, Electron Microscopy, Polymer, Negative Staining, Labeling

Journal: ACS central science

Article Title: Electronic Energy Migration in Microtubules.

doi: 10.1021/acscentsci.2c01114

Figure Lengend Snippet: Figure 4. Average weighted tryptophan fluorescence lifetime of different tubulin polymerization states as a function of AMCA concentration. (A) Free GTP tubulin compared to that of microtubules polymerized using GTP-tubulin, (B), microtubules polymerized using GTP-tubulin to that of those polymerized using GMPCPP-tubulin, (C) microtubules polymerized using GTP-tubulin in the presence and absence of etomidate and isoflurane. Dashed lines and shaded regions represent mean and standard deviation of tryptophan lifetimes in different tubulin polymerization states in the absence of AMCA. p-Values were calculated to determine significance of differences in average weighted lifetimes between GTP microtubules and other tubulin polymerization states (see Figure S10). Error bars represent standard deviation of experiments conducted n = 3 to n = 5 times.

Article Snippet: AMCA labeled tubulin solution was prepared by reconstituting lyophilized AMCA labeled tubulin powder (TL440m; Cytoskeleton Inc., Denver, CO, USA) in BRB80 supplemented with 10% glycerol and 1 mM GTP, to a final concentration of 45 μM tubulin.

Techniques: Fluorescence, Concentration Assay, Standard Deviation

Journal: ACS central science

Article Title: Electronic Energy Migration in Microtubules.

doi: 10.1021/acscentsci.2c01114

Figure Lengend Snippet: Figure 5. Parameters extracted from Stern−Volmer analysis to estimate extent of diffusion . (A) Stern−Volmer plot for fitting different tubulin polymers with the static quenching model as shown in main text. Lines represent line of best fit to data points shown in the scatterplot. Note the use of a logarithmic scale for the x-axis. (B) Diffusion coefficients and (C) diffusion lengths of tryptophan excitation in different tubulin polymerization states. Data are shown in Table S5. The errors associated with (B) are standard errors for the fit to determine the diffusion coefficient. The error bars associated with (C) are calculated after propagating errors from experimental values included in eqs 1 and 2.

Article Snippet: AMCA labeled tubulin solution was prepared by reconstituting lyophilized AMCA labeled tubulin powder (TL440m; Cytoskeleton Inc., Denver, CO, USA) in BRB80 supplemented with 10% glycerol and 1 mM GTP, to a final concentration of 45 μM tubulin.

Techniques: Diffusion-based Assay

Journal: ACS central science

Article Title: Electronic Energy Migration in Microtubules.

doi: 10.1021/acscentsci.2c01114

Figure Lengend Snippet: Figure 6. Theoretical estimation of interactions among tryptophan and tyrosine residues. (A) Crystal structure of a microtubule composed of 31 tubulin dimers stacked vertically showing the dipole moment orientations of representative tryptophan (green; TRP) and tyrosine (cyan; TYR) residues. (B) Distribution of the coupling constant VCoul between TYR-TYR, TRP-TRP and (C), TYR-TRP residues in 31-dimer long microtubule crystal structure. A projection of VCoul with orientation factor (κ2) of 2/3 is represented as solid lines.

Article Snippet: AMCA labeled tubulin solution was prepared by reconstituting lyophilized AMCA labeled tubulin powder (TL440m; Cytoskeleton Inc., Denver, CO, USA) in BRB80 supplemented with 10% glycerol and 1 mM GTP, to a final concentration of 45 μM tubulin.

Techniques: