Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules

J. A. Tuszynski∗ , T. Luchko, E. J. Carpenter and E. Crawford Department of Physics University of Alberta Edmonton, AB, Canada T6G 2J1 jtus@phys.ualberta.ca April 13, 2004

Abstract We present the results of molecular dynamics computations based on the atomic resolution structures of tubulin published as 1TUB and 1JFF in the Protein Data Bank. Values of net charge, spatial charge distribution and Cartesian dipole moment components are obtained for the tubulin alpha-beta heterodimer. Physical consequences of these results and subsequent computations are discussed for microtubules in terms of the effects on test charges, test dipoles, and neighboring microtubules. Our calculations indicate typical distances over which electrostatic effects can be felt by biomolecules, ions, and other microtubules. We also demonstrate the importance of electrostatics in the formation of the microtubule

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lattice and the tubulin-kinesin binding strength. Keywords: tubulin, kinesin, molecular dynamics, microtubules, electrostatics

1

Introduction

Microtubules (MTs) are protein filaments of the cytoskeleton with lengths that vary but commonly reach 5-10µm. They are composed of 9 to 16 protofilaments when self-assembled in vitro and almost exclusively of 13 protofilaments in vivo[1]. These protofilaments are strongly bound internally and are connected via weaker lateral bonds to form a sheet that is wrapped up into a tube in the nucleation process. These cylindrical protein organelles, found in all eukaryotes, are critically involved in a variety of cellular processes including motility, transport and mitosis[2]. Their component protein, tubulin, is composed of two polypeptides of related sequence, designated α and β. In addition to α- and β-tubulin, many microtubules in cells require the related γ-tubulin for nucleation. Two other tubulins, designated δ and , are widespread, although their roles are still uncertain. The general structure of MTs has been well established experimentally. A small difference between the α- and β-monomers of tubulin allows the existence of at least two lattice types. Moving around the MT in a left-handed sense, protofilaments of the A lattice have a vertical shift of 4.92 nm upwards relative to their neighbors. In the B lattice this offset is only 0.92 nm because the α- and β-monomers have switched positions in alternating filaments. This change results in the development of a structural discontinuity in the B lattice known as a seam. At the molecular level tubulin’s roles are highly complex. For example, microtubules undergo cycles of rapid growth and disassembly in a process known as dynamic instability that appears to be critical for microtubule function, es-

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pecially in mitosis. The kinetics of hydrolysis of the GTP bound to β-tubulin is critical for this process by affecting the loss of the so-called lateral cap that stabilizes the microtubule structure[3]. In addition to forming microtubules, tubulin interacts with a large number of associated proteins. Some of these, such as tektin, may play structural roles; others, the so-called microtubule-associated proteins (MAPs) such as tau or MAP2, may stabilize the microtubules, stimulate microtubule assembly and mediate interactions with other proteins. Still others, such as kinesin and dynein, are motor proteins that move cargoes, e.g. vesicles, along microtubules[4]. The precise molecular basis of the properties of tubulin is still not well understood, in part because tubulin’s highly flexible conformation makes it difficult to crystallize. In a major advance in the field, the three-dimensional structure of bovine brain tubulin has been determined by electron crystallography by Nogales et al. in the presence of zinc ions[5, 6]. Once the three-dimensional structure of a protein has become known it is possible to use affinity modeling to predict the structures of related forms of the protein with some degree of accuracy. The crystallographic results were made available through the Protein Data Bank (PDB) (entries: 1TUB and 1JFF) which allowed us to view the 3D atomic resolution structure of tubulin. Each tubulin monomer is composed of more than 400 amino acids and, in spite of their similarity, slight folding differences can be seen. It is worth stressing that several different versions of both the α- and β-monomers exist and are called isotypes[7]. We have applied affinity modeling techniques to a series of some 290 different tubulins, representing αand β-tubulins from animals, plants, fungi and protists, as well as several γ-, δand -tubulins. A summary of this work will be published elsewhere.

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2

Conformational and electrostatic changes due to GTP hydrolysis

Tubulin is believed to exist in two conformational states depending on whether GTP or GDP is found in the E-site. Importantly, when GTP is found in the E-site tubulin’s net charge is reduced by one. Furthermore, it is believed that having GTP in the exchangeable site is necessary for polymerization while GDP in the exchangeable site contributes to depolymerization[3]. To determine conformational changes due to GTP hydrolysis a series of simulations were carried out as described in Section 2.1. 10 coordinate sets per ps where recorded and analyzed for the final 600ps and 300ps of the first and second equilibrium runs of GTP and GDP tubulin. Common indicators of conformational change such as water accessible surface area, radius of gyration and RMS fluctuations from the crystal structure showed little difference between the four runs. Average structures were produced and root-mean-square (RMS) deviations from the crystal structure as well as differences between the average structures were taken. Similar changes were found among runs with the same nucleotide as with different nucleotides suggesting no significant conformational changes were found. Though similar protocols have found conformational changes in proteins before, for example [8], we believe that these results are likely because the annealing runs were too short to allow a sufficient conformational search to occur. Either several longer annealing runs or alternate computational techniques are required to achieve this.

2.1

Computational methods

Molecular dynamics simulations with CHARMM[9] and the CHARMM27 force field[10] were performed using the crystal structure file 1JFF as initial condi-

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tions for the protein structure. Simulated annealing was preformed on structures containing GTP and GDP in the exchangeable (E) site. Following this, equilibrium molecular dynamics was preformed to obtain average structures from which properties were calculated. In order to use 1JFF as the initial conditions for our simulations it was necessary to remove the taxol molecule and zinc ion and add several parts of the amino acid chains that were missing in the crystal structure. Except for the C-termini, the locations of the missing residues were obtained from the 1TUB structure. In addition, we have been able to predict with some confidence the conformations of the C-termini of tubulin since the C-termini of α- and β-tubulin were not resolved in the original electron crystallographic reconstruction of the tubulin molecule due to their flexibility. Our results raise the possibility that the movements of the C-termini of tubulin may play a major role in kinesin motility and that they may have some novel, hitherto unsuspected, roles in ion transport along microtubules especially in the axoplasm of neurons. The C-termini had been previously added to 1TUB using MolMol[11] and were added to the 1JFF structure in the same manner as the other missing residues. Residues 1A-2A, 27A-64A, 436A-451A, 1B-3B, and 435B-455B were added individually by rootmean-square devitation (RMSD) fitting the backbone atoms of five residues adjacent to the missing segments on either side and pasting the missing residues into the new structure. Besides the Mg2+ ion that was part of the crystal structure 4 Na+ , 55 K+ and 6 Cl− ions were added to the system to maintain electro-neutrality and an ion concentration consistent with that of cytoplasm. An additional K+ was added for simulations with GTP in the E-site. These ions were placed at random positions about the fixed protein in a box 105˚ X 65˚ X 125˚ A A A and equilibrated for 2ns using Langevin dynamics with a friction coefficient of

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62ps−1 and periodic boundary conditions (PBC)[12]. The two systems were then hydrated by tiling a box of 3000 pre-equilibrated water molecules and minimized for 400 steps of steepest descent. Simulated annealing was then performed based the protocol outlined in [8]. Our systems were heated to 500K in 40ps and held there for 10ps and then cooled to 200K over 120ps using time steps of 2fs and utilizing SHAKE to maintain rigid bond lengths. The resulting coordinate sets and their subsequent simulations will be referred to as GTP1 and GDP1. A second coordinate set for each system (GTP2 and GDP2) was produced by maintaining the system at 500K for an additional 15ps before cooling. All four coordinate sets were then equilibrated at 300K for 150ps with 1fs time steps without SHAKE. A total of 2.1ns of production simulation was performed between the four systems. GTP1 and GDP1 each were simulated for 600ps while GTP2 and GDP2 were both simulated for 300ps. Coordinate sets were recorded every 0.1ps.

2.2

Electrostatic properties

For self-assembly of MTs to occur individual tubulins must interact via longrange forces (i.e. electrostatic forces) for attraction and alignment to occur. Since tubulin is highly charged and, thus, naturally repulsive to other tubulins the shape of the electric field and how it interacts with its ionic environment is of great importance. To estimate an upper bound for the maximum distance over which tubulin electric fields may have some significant interaction we may look to the critical concentration of Tu-GTP required for MT growth. While the concentration depends on such factors as the use of nucleation seed, a minimal estimate of 5mM[13] may be used. Typically, the Tu-GTP critical concentration in vitro

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must be higher. This corresponds to an average separation between the centers of mass of 150˚ or, between surfaces, of roughly 100˚. We can consider this the A A maximum distance at which tubulin dimers may interact. For orientation-dependent measurements, such as the dipole moment, we have RMSD fit the α-helix and β-sheet portions of the backbone of each coordinate set to 1JFF. Prior to this we have rotated 1JFF structure 26◦ with respect to the protofilament axis (x-axis) such that the y-axis is tangential and the z-axis is inward and perpendicular to the MT surface[14]. Furthermore, unless otherwise noted, all electrostatics calculations have been carried out with a dielectric constant of 1 corresponding to a vacuous environment. Since the force field parameters have been optimized for a dielectric constant of 1 and the simulations performed under such conditions, this produces the most realistic and relevant results for our simulations. Ions are included where noted (Mg2+ at the N-site is considered part of the protein) and water is generally included though explicit waters except where noted. The porcine tubulin sequences of the α- and β-monomers used have a typical net charge of -24e each at pH 7.0. Including the two nucleotides and the bound Mg2+ the net charge of the structure is -53e and -54e for GDP bound and GTP bound tubulin, respectively. Although we have found no significant conformational differences between any of our simulations we will report the electrostatic properties of both GDP bound and GTP bound tubulin separately due to the differences in net charge. Although no obvious conformational differences were found between GTP1,2 and GDP1,2 there was a difference in ionic condensation around the exchangeable site. In both GDP1 and GDP2 a positive ion is tightly bound to the surface of the exchangeable nucleotide analogous to Mg2+ at the non-exchangeable site. In GTP1,2 two positive ions are tightly bound to the surface of the nucleotide.

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Over the length of the runs these bound ions had an average root-mean-square fluctuation (RMSF) of 0.79˚ for GTP1,2 and 0.53˚ for GDP1,2. Also observed A A ˚ in all simulations was a single ion oscillating 5 to 8A from the exchangeable ˚ nucleotides. Not strongly bound, these had an average RMSF value of 5.7A for ˚ GTP1,2 and 4.3A for GDP1,2. While several ions in all of the simulations became bound to the surface, other than around the E-site, only one other site bound ions in all simulations. A single sodium in GDP1,2 and a single potassium in the case of GTP1,2 was found between residues 417 and 426 of the α-monomer. This is a highly charged area of the N-terminal end of the H12 helix. The sodium ions found in GDP1,2 were much more tightly bound with RMSF of 1.2˚ as compared to 5.1˚ for the A A potassium ions in GTP1,2. The distribution of the electrostatic potential on the surface of the protein in the absence of water and ions is shown in Figure 1. Due to the large net charge of tubulin the surface potential is completely negative. However, potential gradients can be seen across the surface along the lateral and longitudinal directions that are important for binding within the MT. Laterally the almost neutral side of the tubulin, Figure 1(f), will be in contact with its negative side, Figure 1(e), of its neighbors. Similarly, the exposed end of the β-monomer, Figure 1(d) is quite negative in comparison to the exposed end of the α-monomer, Figure 1(c), particularly around the E-site. [Figure 1 about here.] The shape of the electric field at large distances is typically discussed in terms of the multipole expansion. Unfortunately, neither the dipole nor quadrupole moment can be uniquely defined for a structure that has a non-zero net charge. However, by using the center-of-mass as our origin we can attempt to quantify the distribution of charge within the molecule and how this may affect long-range 8

interactions. How this shape changes with the addition of ions and the multipole approximation to it can be seen in Figure 2. Table I summarizes the results of our calculations of the tubulin’s net charge, dipole moment from our simulations. Note that the x-direction in Table 1 coincides with the protofilament axis. The α-monomer is in the direction of increasing x values relative to the β-monomer. The y-axis is oriented radially towards the MT center and the z-axis is tangential to the MT surface. [Table 1 about here.] [Figure 2 about here.]

2.3

Dimer-dimer interactions

Protofilament and microtubule dynamics are a result of tubulin dimer-dimer interactions. In order to quantify these interactions a potential energy map was produced by moving one tubulin dimer relative to another (see Figure 3) and calculating the electrostatic and Lennard-Jones interaction energy in the absence of water and ions using CHARMM and a 30˚ cut-off. A [Figure 3 about here.] The mapping of dimer-dimer interactions was done in a planar grid tangent to the MT surface. The grid spacing used was 2˚ with center-of-mass displaceA ments ranging from 0 to 120˚ longitudinally and -80˚ to 80˚ laterally. Due A A A to the symmetry created by using pairs of identical dimers (produced from the average structures of GTP1 and GDP1) it was not necessary to perform calculations with a negative displacement in the longitudinal axis. Rotation of the dimers was not varied with the lateral separation. As a result these calculations may more closely represent protofilament sheets than MTs.

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Figure 3 shows the two interaction maps. Both show strong local minima corresponding to longitudinal bonds, though they are slightly off-center. This is in part due to steric conflicts caused by flexible side chains in an extended conformation. Minimization of the proteins when in close contact removes these conflicts. The most notable change between the two maps occurs in the lateral ˚ bonds. Figure 3(a) shows a local minima at approximately a 10A longitudinal separation of the dimers, corresponding to a B-lattice. A second, broader minima is centered at about 80˚ and is also consistent with a B-lattice. In A ˚ Figure 3(b) the first minima centered at 10A is sterically excluded. Further more, the lateral binding energies are slightly smaller. Estimates of the binding energies from these maps must be considered qualitative due to the lack of solvation. While applying a dielectric constant of 80 to the system does provided a better estimate it is an under estimate and does not account for the very different dielectric constant of protein. This explains differences between our calculations and those of VanBuren et al.[15] and Sept et al.[16]. A further difference between this calculation those of Sept et al. is the lack of a minimum corresponding to an A-lattice. However, in their calculation this minimum was provided by a surface area term that dominated over a potential maximum in the electrostatic energy. The potential maximum they observed agrees with that found in our own calculations.

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Kinesin and tubulin

We have performed MD simulations for kinesin interacting with a tubulin dimer while the system studied was coupled to an external heat bath. This approach was used earlier by Wriggers and Schulten[8] to propose conformational changes for the kinesin head. Tubulin was heated in the absence of water and ions from 0 K to 400 K over 50 ps and then slowly cooled to 200 K over 120 ps in time

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steps of 1 fs. Conformational changes that were found in tubulin have been seen to affect both lateral and longitudinal contacts, providing some insight into the nature of the dynamical instability of microtubules. We have also seen that the hydrolysis state of kinesin affects its binding affinity for tubulin. Figure 4 shows the shape of the binding potential whose depth is approximately 0.5 eV corresponding closely to the ATP hydrolysis energy that drives the process of kinesin’s walk along the filament. Figure 5 shows the electric charge distribution on the motor domain of kinesin that appears complementary to the charges on tubulin. Furthermore, the calculated kinesin binding site of MTs agrees with results from crystallography. (see Figure 6) [Figure 4 about here.] [Figure 5 about here.] [Figure 6 about here.]

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Conclusions

The electrostatic charge of tubulin, although significantly screened by counterions in solution, could affect microtubule assembly by influencing dimer-dimer interactions on relatively short distances (approximately 5 nm) and the kinetics of assembly. This has been recently demonstrated by Sept et al[16] who calculated the electrostatic energy of protofilament-protofilament interactions and concluded from their work that the two types of microtubule lattices (type A and B) correspond to the local energy minima. The dipole moment could play a role in microtubule assembly and other processes also. This could be instrumental in the docking process of molecules to tubulin and in the proper steric configuration of a tubulin dimer as it approaches 11

a microtubule for binding. A tubulin dimer surrounded by water molecules and counter-ions has a dominant dipolar contribution to the electrostatic energy as shown in Figure 2. Once a microtubule has been formed, the greater the dipole of each of its units is, the less stable the microtubule since dipole-dipole interactions provide a positive energy disfavoring a microtubule structure. Note that the strength of the interaction potential is proportional to the square of the dipole moment hence microtubule structures formed from tubulin units with larger dipole moments should be more prone to undergo disassembly catastrophes compared to those microtubules that contain low dipole moment tubulins. In work published elsewhere [17] we have also considered the role of electrostatics in the interactions between tubulin, microtubules and other charged or polarized molecules. In particular, we have shown that in spite of Debye screening, a microtubule can exert a Coulomb force on a charged particle that is up to 5 nm away from its surface. The dipole-dipole forces that have been calculated are negligible for the most part. However, when two microtubules are found in the same vicinity, they can exert significant forces of repulsion even in the presence of ionic screening. This is increased by the negatively charged C-termini protrude perpendicularly to the microtubule, explaining the existence of the so-called ‘zone of exclusion’[2] known to cell biologists for many years. We have shown that the microtubule structure, in particular the lateral binding between protofilaments, is consistent with the location of positive and negative segments of the electrostatic potential for optimal binding. It is worth mentioning that a recent paper[18] showed the electrostatic surface of the whole microtubule following computations involving the Poisson-Boltzmann equation. From these calculations a dramatic difference between the plus and minus ends of a microtubule has been revealed. It is very likely that this difference leads to the well-known difference in polymerization kinetics involving these two ends.

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Finally, the state of the C-termini could mediate how motor proteins such as kinesin bind to and move on microtubules. Our simulations show that kinesin binds preferentially to upright C-termini and not to C-termini lying on the surface of the microtubule. Very minor changes in the local ionic environment or the pH could halt the processive motion of a two-headed kinesin by collapsing the C-termini. One can postulate that the proportion of C-termini that are in the upright conformation in a given portion of the microtubule could determine the actual rate of kinesin movement. It is likely that such arguments could apply to other motor proteins as well. We hope that the new insights gained by performing these computations will be useful in our understanding of the cellular machinery as well as in the efforts to construct nanomachinery that uses biomolecular components or hybrid structures mimicking the effects observed in living cells.

Acknowledgments
This research was supported by grants from NSERC and MITACS-MMPD.

References
[1] D. Chretien and R. Wade, Biol. Cell 71, pp. 161–174, (1991). [2] P. Dustin, Microtubules, Springer-Verlag, Berlin, (1984). [3] H. Flyvbjerg, T. Holy, and S. Leibler, Phys Rev. E. 54(5), pp. 5538–5560, (1996). [4] M. Kikkawa, E. Sablin, Y. Okada, H. Yajima, R. Fletterick, and N. Hirokawa, Nature 411, pp. 439–445, (2001).

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[5] E. Nogales, S. Wolf, and K. Dowling, Nature 391, pp. 199–203, (1998). PDB ID 1TUB. [6] J. L¨we, H. Li, K. Dowling, and E. Nogales, J. Mol. Biol. 313, pp. 1045– o 1057, (2001). PDB ID 1JFF. [7] Q. Lu, G. Moore, C. Walss, and R. Luduena, Structural and functional properties of tubulin isotypes in Advances in Structural Biology, 5, pp. 203– 227, Jai Press, Stanford U.S.A., (1998). [8] W. Wriggers and K. Schulten, Biophysical Journal 75, pp. 646–661, (1998). [9] B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S. Swaminathan, and M. Karplus, J. Comp. Chem 4, pp. 187–217, (1983). [10] A. D. MacKerell, Jr., D. Bashford, M. Bellott, R. Dunbrack Jr., J. Evanseck, M. Field, S. Fischer, J. Gao, H. Guo, S. Ha, D. JosephMcCarthy, L. Kuchnir, K. Kuczera, F. Lau, C. Mattos, S. Michnick, T. Ngo, D. Nguyen, B. Prodhom, W. Reiher, III, B. Roux, M. Schlenkrich, J. Smith, R. Stote, J. Straub, M. Watanabe, J. Wiorkiewicz-Kuczera, D. Yin, and M. Karplus, J. Phys. Chem. B 102, pp. 3586–3616, (1998). [11] R. Koradi, M. Billeter, and K. Wuthrich, J. Mol. Graphics 14, pp. 51–55, (1996). [12] B. Roux, Personal communications (2002). [13] M. Symmons, S. Martin, and P. Bayley, Journal of Cell Science 109, pp. 2755–2766, (1996). [14] H. Li, D. J. DeRosier, W. V. Nicholson, E. Nogales, and K. H. Downing, Structure 10, pp. 1317–1328, (2002).

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[15] V. VanBuren, D. J. Odde, and L. Cassimeris, PNAS 99, pp. 6035–6040, (2002). [16] D. Sept, N. Baker, and J. A. McCammon, Protein Science 12, pp. 2257– 2261, (2003). [17] J. Tuszynski, J. Brown, E. Crawford, E. Carpenter, M. Nip, J. Dixon, and M. Sataric, Mathematical and Computer Modelling , (accepted April 11 2003). [18] N. Baker, D. Sept, S. Joseph, M. Holst, and J. McCammon, Proceedings of the National Academy of Sciences 21, pp. 10037–10041, (2001). [19] W. Humphrey, A. Dalke, and K. Schulten, J. Molecular Graphics 14, pp. 33–38, (1996). http://www.ks.uiuc.edu/Research/vmd/.

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Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules J. A. Tuszynski et al.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 1: Electrostatic potential at the water accessible surface. Blue is negative, white is neutral and red (none shown) is positive. The views are facing radially out of (a) and into (b) the MT, the exposed α-end (c) and the exposed β-end (d) and tangential to the MT with the α monomer on the right(e) and the left (f). Images created with VMD [19].

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Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules J. A. Tuszynski et al.

(a)

(b)

(c)

Figure 2: A tubulin dimer in vacuum (a) is seen principally as a monopole from the point of view of the electrostatic potential while it is mainly dipolar when surrounded by counterions and water ((b) and (c)). Images created with VMD [19].

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Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules J. A. Tuszynski et al.

(a)

(b)

Figure 3: Potential energy tubulin-tubulin interaction map for the average structures of (a) GTP1 and (b) GDP1. The axes represent the center-of-mass displacement between the two dimers while the colour represents the potential energy in kcal/mole. Areas of high steric conflict have been colored blue.

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Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules J. A. Tuszynski et al.

Potential (eV)

Kinesin Position Along Protofilament (Å)

Kinesin Position (Å)

Figure 4: Map of the interaction energy between a kinesin head and a protofilament.

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Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules J. A. Tuszynski et al.

Figure 5: Electrostatic potential at the water accessible surface of the kinesin head domain (PDB ID: 1I6I)[4]. Figure prepared using MolMol[11].

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Results of Molecular Dynamics Computations of the Structural and Electrostatic Properties of Tubulin and Their Consequences for Microtubules J. A. Tuszynski et al.

Figure 6: A kinesin head domain is shown bound to two tubulin monomers. Data from PDB file 1IA0[4]. Image created with VMD [19].

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Table I: The key electrostatic properties of the tubulin dimer with GTP or GDP in the exchangeable site. Tubulin properties (w.r.t. center of mass) charge (electrons) dipole (Debye) overall magnitude x-component y-component z-component Tu-GTP -54 4850 700 200 4800 360 140 180 341 RMSF Tu-GDP -53 5090 870 370 4970 140 220 410 130 RMSF

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