Structural Analysis of DJ-1 Glyoxalase Activity by Mix-and-Inject Serial Synchrotron Crystallography

Authors

Coleman Dolamore, University of Nebraska-LincolnFollow

First Advisor

Mark A. Wilson

Committee Members

Donald Becker, David Berkowitz, Jaekwon Lee, Limei Zhang

Date of this Version

7-2024

Document Type

Thesis

Citation

A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science

Major: Biochemistry

Under the supervision of Professor Mark A. Wilson

Lincoln, Nebraska, July 2024

Comments

Copyright 2024, Coleman Dolamore. Used by permission

Abstract

Observing enzyme structures while they are catalyzing a reaction has been a major goal of both enzymology and structural biology for decades. With the advent of time-resolved serial X-ray crystallography using X-ray free electron lasers (XFELs) and synchrotron sources, enzyme reactions can be monitored in real time in crystallo. Here, we use two different approaches to study related enzymes involved in methylglyoxal detoxification. For human DJ-1, we used pink beam mix-and-inject serial crystallography (MISC) at the Advanced Photon Source (BioCARS 14-ID) to probe the controversial mechanism of DJ-1’s action on methylglyoxal by using serial Laue diffraction. The high flux of the pink X-ray beam permitted the crystals to be exposed to X-rays on the order of microseconds as opposed to millisecond exposure times more common with monochromatic X-rays. Catalytic intermediates were observed at six time points and analysis clarified the structural mechanism for DJ-1 catalysis.

We performed initial methods development to enable a time-resolved microcrystal electron diffraction (micorED) workflow to explore the mechanism of glyoxalase III (Hsp31) from Candida albicans. Hsp31, also a homolog of DJ-1, converts methylglyoxal to lactate with a kcat that is approximately 100x faster than DJ-1. Time-resolved microED requires spraying Hsp31 microcrystals after a precalculated interaction time with methylglyoxal onto a cryo grid before plunge-cooling in liquid ethane and collecting diffraction images using an electron microscope. Our initial efforts show that mix-and-spray techniques may be viable for time-resolved micro electron diffraction studies of enzymes. This exciting technique presents the opportunity to not only assess the differences between Hsp31 and DJ-1 that make Hsp31 ~100x faster than DJ-1, but also to develop a new method of time-resolved crystallography.

Advisor: Mark A. Wilson