X-ray free-electron laser based methods for structural and ultrafast dynamics studies of a photosynthetic reaction centre
Abstract
Life on earth is fuelled by the energy of sunlight, which must first be captured and converted into a chemical energy form useful to the cell. This process is known as photosynthesis and the major pathway of this energy conversion is via photosynthetic reaction centres. These enzymes convert the energy content of an absorbed photon into a transmembrane potential difference via the movements of electrons. Increasing our knowledge of the three-dimensional fold and structural changes that takes place within photosynthetic reaction centres is therefore of considerable importance for understanding biological photosynthesis.
The aim of this work has been to adapt methods for both crystallographic and solution phase structural studies of membrane proteins to the unique properties of X-ray free-electron laser (XFEL) radiation. To accomplish this, a new crystallization technique for the photosynthetic reaction centre from the purple bacterium Blastochloris viridis (RCvir) was developed which was suitable for serial femtosecond crystallography (SFX) experiments at an XFEL. Our initial experiments at the Linac Coherent Light Source (LCLS), the world’s first XFEL, yielded an SFX structure of RCvir to 8.2 Å resolution. After the LCLS decreased the X-ray wavelength at which the facility could operate, and in combination with improved crystallization conditions, we later resolved the SFX structure of RCvir to 3.5 Å resolution.
Whether or not ultrafast structural changes in RCvir occur in photosynthesis has been debated for two decades. We addressed this question by developing time-resolved wide-angle X-ray scattering (TR-WAXS) studies at the LCLS that could capture rapid structural changes in solubilized samples of RCvir. Proof-of-principle experiments revealed a structural deformation that propagated through the RCvir protein following multi-photon absorption by its cofactors, enabling a protein quake through a photosynthetic protein to be visualized. Further insight was provided by a second TR-WAXS experiment in which this structural signal was observed in the data as the pump laser fluence was decreased to less than one photon absorbed per RCvir molecule. This result implies that, even under physiological conditions of normal sunlight, ultrafast protein structural rearrangements may influence the primary charge separation events of biological photosynthesis.
Parts of work
Paper I: Lipidic phase membrane protein serial femtosecond crystallography ::doi::10.1038/nmeth.1867 Paper II: Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography ::doi::10.1038/ncomms3911 Paper III: Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser ::doi::10.1038/nmeth.3067 Paper IV: Ultrafast structural changes in photosynthesis - Manuscript
Degree
Doctor of Philosophy
University
University of Gothenburg. Faculty of Science
Institution
Department of Chemistry and Molecular Biology ; Institutionen för kemi och molekylärbiologi
Disputation
Fredagen den 5 december 2014, kl 9.00, Hörsal Åke Göransson, Medicinaregatan 15C
Date of defence
2014-12-05
david.arnlund@chem.gu.se
david.arnlund@gmail.com
Date
2014-11-14Author
Arnlund, David
Keywords
WAXS
wide-angle x-ray scattering
XFEL
x-ray free-electron laser
reaction center
structural biology
ultrafast dynamics
Publication type
Doctoral thesis
ISBN
978-91-628-9236-4
Language
eng