Date of Award
December 2023
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Physics
First Advisor
Marius Schmidt
Committee Members
Emina A Stojković, Peter Schwander, Valerică Raicu, Ionel Popa
Abstract
X-ray crystallography is a key method in determining the 3-dimensional (3D) structures of proteins at near atomic resolution. A more complete understanding of the protein function requires that structural changes are investigated as they happen during reaction. With time-resolved crystallography (TRX), it is possible to unify protein structure determination with reaction dynamics. A reaction is initiated in a macromolecular crystal, and the resulting change in the structure is investigated by X-ray diffraction. The structures of intermediates that form and decay during the reaction can be determined. Similarly, the fundamental displacements of atoms associated with chemical reactions in proteins can be observed. In the last decade, X-ray free electron laser (XFEL) facilities became available for macromolecular structure determination. X-rays generated by XFELs feature highly brilliant, femtosecond (fs) pulses. Good quality diffraction data can be collected from a microcrystal with a single X-ray pulse that lasts only a few fs. This enormous X-ray intensity destroys the crystal after a single exposure. Due to the ultrashort nature of the X-ray pulses, a diffraction pattern is recorded before the crystal is destroyed. A complete radiation damage free dataset is, therefore, collected from thousands of tiny crystals serially injected into the X-ray interaction region. Since each diffraction is collected from a fresh crystal, reversible and irreversible reactions may be studied equally well at room temperatures. If the protein is naturally photoactive, the reaction can be initiated (pumped) by exposing the crystal to a laser light pulse with an appropriate wavelength. After a delay (that can be femtoseconds or longer) the resulting change is probed by an X-ray pulse. For non-photosensitive proteins a more general method to trigger a reaction is required. Mix-and-inject serial crystallography (MISC) was developed to explore irreversible enzymatic reactions. Microcrystals are mixed with a substrate before injecting the mixture into the X-rays. The diffusion of the substrate into the active sites of the enzymes inside the crystal starts the reaction. The resulting structural changes are investigated by X-rays. In this dissertation results from four projects are described that involve enzymes suitable for time-resolved structure determination. In the first project MISC is applied to understand the inhibition of the Mycobacterium tuberculosis beta(β) lactamase enzyme (BlaC) by a suicide inhibitor. Here, the β-lactam inhibitor sulbactam is used and mixed with BlaC microcrystals. From a time series of MISC data spanning from 3 ms to 700 ms, ligand gating and tunneling, cooperativity, induced fit and conformational selection mechanisms are observed and described with near-atomic precision. The second project aims at the determination of the structure of the main protease (Mpro) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at room temperature. This structure was determined at an XFEL during the COVID-19 pandemic. It will be a prerequisite for future MISC experiments. In a third project, early events of signal propagation in a photoactive enzyme called phytochrome are investigated. X-ray structures were determined at an XFEL on picosecond (ps) time-scales using the pump probe technique. A reaction is started with a 640 nm laser light pulse. The ps structural changes show how the central chromophore responds to photon absorption. Large flexible proteins are difficult to crystallize and are unsuitable for investigation with crystallography. In these cases, structures can be determined using single particle cryo-EM. Here, the structure of a full length intact phytochrome has been determined with cryo-EM. This structure is the starting point for future, ground-breaking time-resolved experiments with cryo-EM.
Recommended Citation
Malla, Tek Narsingh, "TIME-RESOLVED STRUCTURAL INVESTIGATIONS ON BIOLOGICAL MACROMOLECULES" (2023). Theses and Dissertations. 3421.
https://dc.uwm.edu/etd/3421