Khatooni, Zahed (2021) Study of cations effect on microtubule nucleation. Doctoral (PhD) thesis, Memorial University of Newfoundland.
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Abstract
Microtubules (MT) are nanoscale-sized filaments actively involved in the segregation of chromosomes, the movement of vesicles, and the maintenance of eukaryotic cell morphology. The α, β-tubulin heterodimer is the building block of MTs, and γ-tubulin is the critical protein of the ring complex for MTs nucleation through direct interactions with the α, β-tubulin heterodimers. Molecular Dynamics (MD) simulation of GDP and GTP bound γ-tubulin monomer and dimer have been carried out in the presence of NaCl, MgCl₂, and ZnCl₂ as electrolytes. In this study, the Mg⁺², Zn⁺², and Na⁺ effects on γ-tubulin conformation in GTP and GDP liganded and its unliganded monomer and dimer have been investigated. It has been found that both liganded and unliganded γ-tubulin monomer and dimer adapt curved and intermediate conformations relative to β-tubulin straight conformation. The lattice model, which describes the role of lateral interactions in inducing the α, β-tubulin straightness, is an appropriate model for γ-tubulin dimers and monomers compared to the allosteric model. Functional motions were observed for secondary structural segments of H2-H3 helices, M, and T5 loops, as well as H6-H7 segment of the N-terminal and the intermediate domains of the γ-tubulin. Also, H11 and H12 helices belonging to the C-terminal domain of γ-tubulin have been shown to contribute to the biological function. At the GTP binding site, the existing Mg⁺² compared to the Zn⁺² and Na⁺ contribute to stronger electrostatic and L-J interactions between residues of the binding site and nucleotides, either GTP or GDP. Residues of the GTP binding site that are interacting with oxygen atoms of α-γ phosphate, including Gln12, Cys13, Gly144, Thr145, and Gly146, exhibit stronger affinities for GTP and GDP compared to other residues in simulations. This MD result is consistent with wet-lab experiments in other organisms, such as yeast γ-tubulin. The exact molecular position of the high-affinity metal-binding site, which plays a role in the function and stability of tubulins, was shown to be the oxygen atoms of β-γ phosphate of the GTP and GDP β phosphate in the binding site. In conclusion: despite the same sequence and structural similarity, γ-tubulin adapts different conformations as compared to α or β tubulin. The lattice model appeared to better explain the straightness process compared to the allosteric model. The Mg⁺² cations, due to their interactions, play a major role in γ-tubulin atomistic behavior compared to Na⁺ and Zn⁺².
| Item Type: | Thesis (Doctoral (PhD)) |
|---|---|
| URI: | http://research.library.mun.ca/id/eprint/15889 |
| Item ID: | 15889 |
| Additional Information: | Includes bibliographical references -- Restricted until December 1, 2023 |
| Keywords: | microtubule nucleation, gamma-tubulin, cations, MD simulation, GTP - GDP |
| Department(s): | Pharmacy, School of |
| Date: | January 2021 |
| Date Type: | Submission |
| Digital Object Identifier (DOI): | https://doi.org/10.48336/B1VV-P744 |
| Medical Subject Heading: | Microtubules; Binding Sites: Phosphates; Tubulin |
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