Date of Award

December 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physics

First Advisor

Marius Schmidt

Committee Members

Abbas Ourmazd, Ionel Popa, Peter Schwander, Arsenio Pacheco

Keywords

bacterial phytochrome, photoactive yellow protein, Beta-lactamases, Data analysis at XFELs, pump-probe and mix and inject crystallography, Time-resolved serial femtosecond crystallography (TRSFX), X-ray free electron lasers, X-ray protein crystallography

Abstract

Time resolved serial femtosecond crystallography (TR-SFX) utilizes X-ray crystallography to visualize the reaction of molecules in real time at the atomic level. Crystals of biological macromolecules are exposed to powerful X-ray pulses. The X-ray radiation emitted by the crystal is then measured by an X-ray sensitive area detector that produces an image called a diffraction pattern. These patterns are analyzed to determine a three-dimensional atomic structure of the biological macromolecule.The ultimate goal of TR-SFX is to make a “molecular movie” that shows the reaction dynamics of a biological process. For this, a reaction is started in a macromolecular crystal and its three-dimensional atomic structures at different time intervals are determined. When these structures are played in a timely order, a molecular movie is recorded. A perfect analogy of this in a real life would be taking pictures of someone dancing, and playing the succession of pictures to observe the dance. TR-SFX is a method that requires an X-ray Free Electron Laser (XFEL). The XFEL produces X-ray pulses of tens of femtosecond duration with 1012 photons per pulse. These pulses are so strong that the crystals are destroyed after being exposed to a single pulse. Since the X-ray pulses ii are so short, diffraction patterns are collected by the detector just before the crystals are destroyed which is called “diffraction before destruction”. Most impressively, at XFELs, each diffraction pattern is obtained from a fresh crystal. As a result, both reversible and non-reversible reactions are placed in an equal footing and can be studied similarly. Pump-probe TR-SFX and mix and inject crystallography (MISC) are two cornerstones of TR-SFX. In pump-probe TR-SFX, photoactive macromolecules within the crystals are activated using an optical laser called the pump, and the reaction is probed by XFEL pulses. Whereas in MISC, biomolecular crystals are mixed with a substrate, and the structural changes are probed by XFEL pulses in a time resolved fashion. This dissertation presents the time-resolved studies of an enzyme called b-lactamase (BlaC) and two photoactive proteins - photoactive yellow protein (PYP) and phytochromes. MISC was used for the study of the enzymatic reaction of BlaC with an antibiotic called Ceftriaxone (CEF). This proof-of-principle experiment conducted at Linac Coherent Light Source (LCLS) operated by Stanford University in Menlo Park, California, showed how CEF binds at the active site of BlaC. Similarly, a pump-probe experiment on PYP was accomplished at the first megahertz XFEL, European XFEL (EuXFEL), in Hamburg, Germany. This experiment was performed to explore the picosecond regime of the photocycle of PYP and test the feasibility of TR-SFX at high repetition rates XFEL. Finally, another pump-probe experiment on phytochromes was conducted at Spring-8 Angstrom Compact free electron Laser (SACLA), in Harima, Japan. With this experiment, we have observed the Z-E isomerization of phytochromes for the first time and determined the previously unknown time-resolved structure at 1ps. iii

This dissertation also presents data acquisition techniques and processing methods used at XFELs. It explains methods to analyze millions of patterns obtained during an experiment, with the goal of solving the X-ray structures of different intermediates of the reaction. In summary, my dissertation explains everything from the beginning of the experiment to the production of a molecular movie.

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