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Time Resolved Diffraction Studies of Structural Changes in Sensory Rhodopsin

Doktorsavhandling
Författare Robert Bosman
Datum för examination 2019-12-18
ISBN 9789178337378
Förlag Göteborgs universitet
Publiceringsår 2019
Publicerad vid Institutionen för kemi och molekylärbiologi
Språk en
Länkar hdl.handle.net/2077/62253
Ämnesord Rhodopsin, SRII, SRII-HtrII, Time-resolved crystallography, Time-resolved wide angle x-ray scattering, TR-X, TR-WAXS, Serial crystallography
Ämneskategorier Kemi

Sammanfattning

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.

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