2026-09-18T17:58:16Zhttps://gupea.ub.gu.se/server/oai/request

oai:gupea.ub.gu.se:2077/622532019-12-14T02:36:09Zcom_2077_29047com_2077_4716com_2077_10556col_2077_29054col_2077_10557

Bosman, Robert 2019-11-29T07:51:06Z 2019-11-29T07:51:06Z 2019-11-29 9789178337378 http://hdl.handle.net/2077/62253 Responding to different light conditions is an essential process for many organisms on earth. Unicellular organisms are no exception to this and mechanisms for controlling cellular movement must often be sensitive to light. Light sensing proteins commonly have internally bound chromophores that, when activated by specific light wavelengths, propagate structural changes through a protein to produce an appropriate cellular response. Microbial rhodopsins are a family of transmembrane proteins that harness light to perform a range of functions. These rhodopsins have been found to act as ion pumps, channels and light sensing proteins. They all utilize similar chemistry through a covalently bound retinal to perform these diverse functions. In this thesis, time-resolved structural techniques are utilized to track the changes in sensory rhodopsin II (SRII) a photophobic blue-light sensor in archaea that protects the cell against harmful UV-radiation. SRII is bound in the membrane to a transducer protein (HtrII) that extends into the cell to affect a response. Time-resolved structural biology has undergone a period of rapid methodological development. Inspired by the data collection challenges presented by X-ray free electron lasers (XFELS), serial crystallography has proved remarkably effective in resolving protein dynamics in crystals by time-resolved studies. These methods have more recently been used at synchrotrons. Recent work has shown that time-resolved serial millisecond crystallography (TR-SMX) on membrane protein microcrystals growing in lipidic cubic phase (LCP) is possible at synchrotrons. This complements time- resolved X-ray solution scattering (TR-XSS) methods already employed at synchrotron sources to measure protein dynamics. In this thesis, we utilize both methods to gain new insight into SRII and SRII-HtrII dynamics and structure. The papers presented here outline new crystallization conditions for SRII and SRII-HtrII that do not require lipid reconstitution. At the Swiss Light Source, we measured a light-activated structure for SRII that provides a istructural explanation of the long-lived signalling states using TR-SMX. We also collected a low-resolution room temperature SRII-HtrII structure that reveals new features and which paves the way for time-resolved serial femtosecond crystallography (TR-SFX) measurements at XFELs., Solution X- ray scattering experiments were carried out on SRII and SRII-HtrII to observe complex dynamics. These revealed that the presence of transducer inhibits the EF-helix motion, providing evidence that this motion in involved in signal transduction. eng Well-based Crystallization of Lipidic Cubic Phase Microcrystals for Serial X-ray Crystallography Experiments. https://doi.org/10.1107/S2059798319012695 Retinal Isomerization in Bacteriorhodopsin Captured by a Femtosecond X-ray Laser. https://doi.org/10.1126/science.aat0094 Structural explanation for the prolonged photocycle in Sensory Rhodopsin II revealed by room temperature serial millisecond crystallography. Manuscript. Serial millisecond crystallography structure of the Sensory Rhodopsin II: transducer complex. Manuscript. Time resolved x-ray scattering observations of light induced structural changes in Sensory Rhodopsin II. Manuscript. Rhodopsin SRII SRII-HtrII Time-resolved crystallography Time-resolved wide angle x-ray scattering TR-X TR-WAXS Serial crystallography Time Resolved Diffraction Studies of Structural Changes in Sensory Rhodopsin Text Doctoral thesis Doctor of Philosophy robert.bosman@gu.se University of Gothenburg. Faculty of Science Karl Isaksson 2019-12-18 MNF open.access