The equilibrium assembly of purified GDP-tubulin into microtubules induced by taxol and Taxotere has been studied as a function of solution variables, ligand, and nucleotide, in 10 mM sodium phosphate buffers. Assembly is coupled to the binding of one taxoid molecule per tubulin heterodimer, while binding to the unassembled protein is not detected within ligand solubility limits. Linked functions analysis has indicated that two Mg2+ and no more H+ ions are bound per tubulin-taxoid polymerized, and the heat capacity change is negligible within experimental error (determined by van't Hoff analysis and by differential scanning calorimetry), in contrast with drug-free control microtubule assembly and with the abnormal polymerization of the tubulin-colchicine complex. The apparent enthalpy change is ca. 240 kJ mol-1 (calorimetry), and the process is entropy driven. The apparent standard free energy change of taxoid-induced elongation at 2 mM free Mg2+, pH 6.1-6.7, and 37 degrees C is -29.5 +/- 0.4 (taxol) or -31.5 +/- 0.4 kJ mol-1 (Taxotere). This is independent of taxoid excess, which has indicated that the process measured corresponds to the elongation equilibrium of the fully liganded protein. Comparison to elongation in the absence of drug suggests an apparent linkage free energy change of binding and polymerization of -11.3 +/- 1.2 kJ mol-1. The taxoid-induced elongation of GTP-tubulin proceeds with an increment of apparent free energy change of -2.5 +/- 0.4 kJ mol-1 over GDP-tubulin. It is proposed that the taxoid binding changes the conformation of GDP-tubulin from inactive to active, allowing productive binding and elongation at the microtubule end. Among several possible model mechanisms discussed, it is particularly attractive to think of taxoids as double-sided ligands, which bind to tubulin at the microtubule end and participate in a lateral contact interface with the newly added tubulin molecule. In the kinetic pathway of assembly, these ligands should bind first to inactive Mg(2+)-induced linear GDP-tubulin oligomers and transform them into active bidimensional polymerization nuclei. Study holds ProTherm entries: 4597 Extra Details: GDP(1 mM) and ethylenediaminetetraacetic acid(1 mM) were added in the experiment taxoid molecule; tubulin-colchicine complex; polymerization;,kinetic pathway
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||2|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MYTFVVRDENSSVYAEVSRLLLATGHWKRLRRDNPRFNLMLGERNRLPFGRLGHEPGLMQLVNYYRGADKLCRKASLVKLIKTSPELAESCTWFPESYVIYPTNLKTPVAPAQDGIHPPLHSSRTDEREFFLASYNRKKEEGEGNVWIAKSSAGAKGEGILISSDATELLDFIDNQGQVHVIQKYLERPLLLEPGHRKFDIRSWVLVDHQFNIYLYREGVLRTASEPYHMDNFQDKTCHLTNHCIQKEYSKNYGKYEEGNEMFFEAFNRYLTSALNITLESSILLQIKHIIRSCLMSVEPAISTKHLPYQSFQLFGFDFMVDEELKVWLIEVNGAPACAQKLYAELCQGIVDIAIASVFPPPDAEQQPPQPATFIKL|