Using genetic code expansion to investigate the functional dynamics of proteins
Final Report Abstract
This project has contributed to transform genetic code expansion technology from an engineering discipline to a standard technology in biochemical and cell biological research. Our efforts to optimize the incorporation efficiency of two distinct unnatural amino acids resulted in established expertise to produce FRET-labelled proteins, which can be used to study protein dynamics in vitro. We have begun to use this technology for the investigation of importin- conformational dynamics in response to increased crowding of the solution. Using UV-activatable crosslinker amino acids, we have obtained insights into the mechanistic principles of chromosome condensation in mitosis that would not have been attainable by other means. We subsequently expanded our approach to histone-histone chaperone interactions in living yeast and are now exploring the combination of in vivo crosslinking with quantitative proteomics to survey the changes in the interactome of the nucleosome from interphase to mitosis.
Publications
- (2011) The H2B ubiquitin ligase RNF40 cooperates with SUPT16H to induce dynamic changes in chromatin structure during DNA double-strand break repair. Cell Cycle 10, 3495-3504
Kari, V., Shchebet, A., Neumann, H., and Johnsen, S. A.
- (2014) A cascade of histone modifications induces chromatin condensation in mitosis. Science 343, 77-80
Wilkins, B. J., Rall, N. A., Ostwal, Y., Kruitwagen, T., Hiragami-Hamada, K., Winkler, M., Barral, Y., Fischle, W., and Neumann, H.
(See online at https://doi.org/10.1126/science.1244508) - (2014). Optimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes. Chembiochem 15, 1800-1804
Lammers, C., Hahn, L.E., and Neumann, H.
(See online at https://doi.org/10.1002/cbic.201402033) - (2015) Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation. eLife
Kruitwagen, T., Denoth-Lippuner, A., Wilkins, B. J., Neumann, H. and Barral, Y.
(See online at https://doi.org/10.7554/eLife.10396) - (2015) Genetically Encoding Lysine Modifications on Histone H4. ACS Chem Biol 10, 939-44
Wilkins, B.J., Hahn, L.E., Heitmuller, S., Frauendorf, H., Valerius, O., Braus, G.H., and Neumann, H.
(See online at https://doi.org/10.1021/cb501011v) - (2015) In vivo mapping of FACT-Histone interactions identifies a role of Pob3 C-terminus in H2A-H2B binding. ACS Chem Biol 10, 2753-63
Hoffmann, C. and Neumann, H.
(See online at https://doi.org/10.1021/acschembio.5b00493) - (2015) MD Simulations and FRET Reveal an Environment-Sensitive Conformational Plasticity of Importin-β. Biophys J 109, 277-86
Halder, K., Doelker, N., Van, Q., Gregor, I., Dickmanns, A., Baade, I., Kehlenbach, R.H., Ficner, R., Enderlein, J., Grubmueller, H., Neumann, H.
(See online at https://doi.org/10.1016/j.bpj.2015.06.014) - (2016) Chromosome condensation and decondensation during mitosis. Curr Opin Cell Biol 40, 15-22
Antonin, W. and Neumann, H.
(See online at https://doi.org/10.1016/j.ceb.2016.01.013) - (2016) The use of unnatural amino acids to study and engineer protein function. Curr Opin Struct Biol 38, 119-128
Neumann-Staubitz, P. and Neumann, H.
(See online at https://doi.org/10.1016/j.sbi.2016.06.006) - (2018) Trapping Chromatin Interacting Proteins with Genetically Encoded, UV-Activatable Crosslinkers In Vivo. Methods Mol Biol, 1728, 247-262
Hoffmann, C., Neumann, H., Neumann-Staubitz, P.
(See online at https://doi.org/10.1007/978-1-4939-7574-7_16)
