Exposure of beta-tubulin regions defined by antibodies on an Arabidopsis thaliana microtubule protofilament model and in the cells

<p>Abstract</p> <p>Background</p> <p>The function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited.</p> <p>Results</p> <p>Using homology modeling we have generated an <it>Arabidopsis thaliana </it>microtubule protofilament model that served for the prediction of surface exposure of five β-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (β1-10), TUB2.1 (β426-435) and TU-14 (β436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of β-tubulins (behind β425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that β-tubulins could be one of the targets for tyrosine kinases.</p> <p>Conclusions</p> <p>We predicted surface exposure of five β-tubulin epitopes, as well as tyrosine residues, on the surface of <it>A. thaliana </it>microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.</p> <p>The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.</p

Exploring potential energy surfaces in ground- and excited states

Chemical reactivity of atoms, molecules and ions is governed by their underlying potential energy surface. Calculating the whole potential energy surface within reasonable bounds, is impossible for all but the smallest molecules. Usually, only parts of the full potential energy surface can be studied, namely stationary points and the minimum energy paths connecting them. By comparing energies of stationary points and their separating barriers, conclusions regarding possible reactions mechanism, or their infeasibility, can be drawn. Taking excited states into account leads to further complications, as now multiple potential energy surfaces have to be considered and root flips between different excited states may occur, requiring effective state-tracking. Part II of this thesis describes the required methods to locate stationary points and minimum energy paths on potential energy surfaces, by using surface-walking, chain-of-states optimization and intrinsic reaction coordinate integration. Several approaches to state-tracking are presented in chapter 4. Results of this thesis are presented in Part III, containing two contributions to the field of photochemistry: chapter 12 provides a possible excited-state reaction mechanism for a biaryl cross-coupling reaction and offers a plausible explanation for its high regioselectivity. The second contribution is the development pysisyphus (chapter 13), an external optimizer implemented in python, aware of excited states and thus the core of this thesis. By implementing the state-tracking algorithms outlined in chapter 4 it allows effective and efficient optimizations of stationary points in ground- and excited-states. The performance of pysisyphus is verified for several established benchmark sets. Results for several excited-state optimizations are presented in section 13.3, where pysisyphus shows good performance for the optimization of sizeable transition-metal complexes