The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice [published erratum appears in J Cell Biol 1995 Apr;129(2):549]

Abstract

Abstract. The standard free energy for hydrolysis of the GTP analogue guanylyl-(a,b)-methylenediphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCPmicrotubules. It was possible to study GMPCPPmicrotubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the halftime for GMPCPP hydrolysis from >10 h to ",,10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s -1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.T HE hallmark for energy-transducing systems such as myosin (Bagshaw and Trentham, 1973), ion-transporting ATPases (Taniguchi, and Post, 1975;Pickart and Jencks, 1984), the mitochondrial ATPase (Grubmeyer et al., 1982) and chloroplast coupling factor (Feldman and Sigman, 1982) is a near-zero free energy for hydrolysis of bound nucleotide triphosphate (NTP)L Negligible free energy is released during NTP hydrolysis since this is stored in the protein conformation until useful work can be done. We have used the hydrolyzable GTP analogue GMPCPP, which contains a methylene linkage between the alpha and beta phosphates, to determine the free energy for hydrolysis of microtubule-bound NTP. We found that the majority of this free energy is stored in the microtubule lattice, so that the standard free energy for hydrolysis is near zero. This stored energy can do work, as in the NTP-independent movement of chromosomes in a reaction coupled to microtubule disassembly (Koshland et al., 1988;Coue et al., 1991 bules parallels the behavior of several energy-transducing systems.In addition to these equilibrium studies, we have characterized the dynamics of microtubules formed with GMPCPP. The recent observation that this substance is not hydrolyzed in microtubules (Hyman et al., 1992) suggested its use in determining the role of the gamma phosphate moiety in microtubule dynamics. Our rationale was that if conditions could be found for forming microtubules containing GMPCP, it would be possible to determine the kinetic behavior of microtubules with a cognate pair of bound nucleotide tri-and diphosphates. Such a comparison was not possible with GTP analo...

Estimates of lateral and longitudinal bond energies within the microtubule lattice

“…Prediction of strong longitudinal bonds and relatively weak lateral bonds is in agreement with structural observations (18). We also estimated that the potential mechanical energy of tubulin-GDP conformational stress in the MT lattice is 2.1-2.5 k B T, in approximate agreement with thermodynamic studies that suggest Ϸ2.8 k B T per tubulin-GDP is stored in the MT lattice (14,22,23).…”

Energetics of the Cooperative Assembly of Cell Division Protein FtsZ and the Nucleotide Hydrolysis Switch

“…The changes in accessible surface area calculated from the data, ⌬ASA apolar ϭ Ϫ2050 Ϯ 280 Å 2 and ⌬ASA pol ϭ Ϫ520 Ϯ 310 Å 2 per FtsZ monomer added to the polymer, are nearly accounted for by the values calculated for the axial contact only in a tubulin-like FtsZ protofilament model (21,25), ⌬ASA apolar ϭ Ϫ1390 Å 2 and ⌬ASA pol ϭ Ϫ980 Å 2 per monomer. The assembly of FtsZ with GMPCPP proceeds at lower critical concentration than with GTP, as for tubulin (49,51,52). The effect of pH was also similar to tubulin (33), with the uptake of less than one H ϩ ion per FtsZ assembled (Fig.…”

“…On the other hand, the difference between GDP-and GTP-liganded FtsZ may be regarded as a shift in the equilibrium between assemblyinactive and active states of the protein (61) in terms of linked equilibria (45), instead of an all or none activation switch. 4 In the cytosol of M. jannaschii, the apparent affinity of FtsZ 4 Assembly of tubulin with the GDP analogue GMPCP has been reported to proceed with critical concentration values that can be estimated to be two orders of magnitude larger than with GMPCPP under the same solution conditions (52,49). Consequently, polymerization of GDP-tubulin might be expected to take place at practically unachievable concentrations of hundreds of mg per ml.…”

Electromagnetic Field of Microtubules: Effects on Transfer of Mass Particles and Electrons

“…Vibrations in the structure generate an oscillating electric field around it. Energy is supplied to the microtubule structure by hydrolysis of GTP to GDP in β tubulins [45,46] In the interphase (a) microtubules display dynamic instability (the growth and the shrinkage of their body indicated by arrows). In the M phase treadmilling causes polymerization from one side (from the center indicated by arrows) and depolymerization from the other side (at the poles of the mitotic spindle).…”