title: Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.
abstract: Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly.
authors.affiliation: Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216, USA.
authors.forename: B
authors.initials: B
authors.lastname: Vulevic
authors.forename: J J
authors.initials: JJ
authors.lastname: Correia
mesh_terms: D000818:Animals; D003078:Colchicine / Q000494:pharmacology; D003198:Computer Simulation; D019281:Dimerization; D006150:Guanine Nucleotides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008854:Microscopy, Electron; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D009010:Monte Carlo Method; D009391:Nephelometry and Turbidimetry; D017239:Paclitaxel / Q000494:pharmacology; D011487:Protein Conformation; D013552:Swine; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D006150:Guanine Nucleotides; D007202:Indicators and Reagents; D014404:Tubulin; D006153:Guanosine Diphosphate; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; D017239:Paclitaxel; D003078:Colchicine