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Time-resolved crystallography on the structural dynamics of cryptochromes and photolyases
Photolyases and cryptochromes form a superfamily (PCSf) of light-directed proteins found in all areas of life. Photolyases repair UV-induced DNA lesions, namely either cyclobutane-pyrimidine dimers (CPD) or pyrimidine-(6-4)-pyrimidone photoproducts, (6-4)PP. The cryptochromes have mostly lost DNA re...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2023
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| Zusammenfassung: | Photolyases and cryptochromes form a superfamily (PCSf) of light-directed proteins found in all areas of life. Photolyases repair UV-induced DNA lesions, namely either cyclobutane-pyrimidine dimers (CPD) or pyrimidine-(6-4)-pyrimidone photoproducts, (6-4)PP. The cryptochromes have mostly lost DNA repair functionality and act as signaling proteins coupling various biological responses to light input. Photolyases and cryptochromes have close evolutionary relationships sharing both a common topology, including the same photoactive regions (FAD, antenna cofactors). During photoactivation, FAD can capture one or two electrons and exist in four possible redox states: oxidized (FADox), anionic semiquinone FAD radical (FAD•−), radical semi-reduced (semiquinone, FADH•) and fully reduced (hydroquinone, FADH−). When blue light absorption drives first electron transfer to the inactive FADox, resulting in a short-lived FAD•− which is intermediate of long-lived protonated FADH•. Furthermore, the second electron transfer leads to the fully photoactivated state, FADH−, which catalyze DNA repair of CPD or (6-4)PP by injecting an electron from excited FADH−* onto the DNA lesion. This study focuses on the structural changes via time-resolved serial femtosecond X-ray crystallography (TR-SFX) to describe how cryptochromes perform signal transduction mechanism in animal-like cryptochrome from the green algae Chlamydomonas reinhardtii (CraCRY). However, CraCRY is not only a cryptochrome, but also has (6-4) photolyase function, meaning (6-4)PP repair. In CraCRY, during first light-driven cycle, three tryptophans and one tyrosine (Y373) act as an electron transfer chain, with Y373 donating the electron to the FAD to produce short-lived radical pair (RP) FAD•−/Y373•+, which evolves to the long-lived FADH•/Y373• RP. The tyrosyl radical is triggering a structural change in the region between the loop carrying the tyrosyl radical and the C-terminal α22- helix, and c-terminal unfolding afterwards. In the photoreduction of CraCRY, 19 snapshot structures were obtained, which showed that signal transduction occurred in three different phases in ns to ms: (1) stabilization of FAD•− and Tyr•+ radicals (ns), (2) stabilization of FAD•− via proton transfer (μs to ms), and (3) c-terminal unfolding (ms to s). These results demonstrated a detailed molecular mechanism for cryptochrome photoactivation. For (6-4)PP repair, we succeeded to preliminarily characterize via in crystallo cryo-trapping of post-illumination intermediates, immediately followed by data collection. We were able to determine three stages of complex dissociation at atomic resolution: (1) base relaxation within the active site, (2) base return towards the unpaired bubble and (3) DNA release from the (6-4)-photolyase active site. For further investigation of photoreduction, mutants of each relevant actor in the signaling state were characterized both spectroscopically and crystallographically. These mutations influenced the formation of the signaling state characterizing the role of individual amino-acids during the CraCRY photocycle. For repairing DNA lesion, (6-4)PP repair mechanism is still not clear. Future TR-SFX experiments will show the whole repair mechanism for (6-4)PP repair. |
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| DOI: | 10.17192/z2023.0521 |