8453 uv vis

MjCobT was dialyzed against 50 mM Na/K phosphate (pH 7.5) and 100 mM NaCl and diluted to a final concentration of 1 mg/ml. Quantification was performed on an Agilent 8453 UV/vis and used an ε₂₈₀ of 20400 M⁻¹ cm⁻¹ as determined by ProtParam^{34 (link)}. The protein sample was loaded with the dialysis buffer to a final concentration of 1 mg/ml before being loaded into cells with 12 mm double-sector Epon centerpieces. The loaded cells were allowed to equilibrate in the rotor for 1 h at 20 °C. Sedimentation velocity data were collected at 50,000 rpm at 20 °C in an Optima XLA analytical ultracentrifuge. Data were recorded at 280 nm in radial step sizes of 0.003 cm. SEDNTERP³⁵ was used to calculate the partial specific volume of MjCobT (0.756018 mL/g) as well as^{36 35 35} the density (1.0093 g/mL) and viscosity coefficient (0.010291) of the buffer. SEDFIT^{37 (link)} was used to analyze the raw sedimentation data. Data were modeled as a continuous sedimentation coefficient distribution [c(s)], and were fit using the baseline, meniscus, frictional coefficient, and systematic time-invariant and radial-invariant noise. Fit data for the experiment had a root mean square deviations of (r.m.s.d) < 0.004 AU. The theoretical sedimentation coefficient (s) values were calculated from the atomic coordinates of MjCobT(PDB ID 3L0Z) using HYDROPRO20^{38 (link)}.

All reagents and solvents were purchased from Sigma-Aldrich, VWR, or Fisher Scientific. Reagents for cellular assays were purchased from ATCC, Invitrogen-Gibco, or VWR. The tubulin polymerization assay kit was purchased from Cytoskeleton Inc. ¹H NMR spectra and ¹³C spectra were recorded at room temperature on a Bruker 400 MHz Ultrashield Plus. Proton and Carbon chemical shifts are expressed in parts per million (ppm, δ scale) and are referenced to the solvent peak. UV-Vis experiments were carried out on either NanoDrop ND-1000 or an Agilent 8453 UV/Vis. Cell Titer Blue (CTB) fluorescence was measured on BioTek Synergy H1 Hybrid plate reader. The UV lamp used in all studies consisted of a simple aquarium light fixture containing two Philips PL-S 9W/2P BLB bulbs. The light was placed at distance of 5 cm from the top of plate. The intensity was measured to be 4.4mW/cm².