Determining the Enzymatic Mechanism of DJ-1 Using a New Approach to Mix-and-Inject Synchrotron Serial Crystallography
Category
Published on
Type
journal-article
Author
Kara Zielinski and Cole Dolamore and Kevin Dalton and Doeke Hekstra and Robert Henning and Vukica Srajer and Mark Wilson and Lois Pollack
Citation
Zielinski, K., Dolamore, C., Dalton, K., Hekstra, D., Henning, R., Srajer, V., Wilson, M., & Pollack, L. (2025). Determining the Enzymatic Mechanism of DJ-1 Using a New Approach to Mix-and-Inject Synchrotron Serial Crystallography. Structural Dynamics, 12(2_Supplement), A116–A116. https://doi.org/10.1063/4.0000425
Abstract
Mix-and-inject serial crystallography (MISC) is a well established technique at X-ray Free Electron Lasers (XFELs) to capture snapshots of the progress of a reaction, typically between a protein and a ligand. Key to this method are specialized diffusive mixers which efficiently mix small molecules into protein crystals for reaction initiation. Given the broad applicability of MISC to essential biological questions, like enzymatic mechanisms and inhibitor design, there is significant interest in adapting it to synchrotrons due to the greater availability of beamtime. Here, we present a new microfluidic device that couples a diffusive mixer to an X-ray compatible observation region for MISC experiments at synchrotrons. Timepoints ranging from ∼20 ms to 30 s are reachable by changing sample and ligand flowrates or the position of the X-ray beam relative to the tip of the mixer. Our first experiments were performed at the BioCARS 14-ID beamline (Advanced Photon Source). Datasets were collected at a 10 Hz repetition rate in about 1–4 hours, due to greater information content of polychromatic images (available at 14-ID) relative to monochromatic diffraction patterns. We studied the enzymatic activity of DJ-1, an important protein in oxidative stress response. Interestingly, DJ-1's enzymatic function has been heavily disputed, but we directly observed DJ-1 acting on one of its purported substrates, methylglyoxal, confirming its role as a glyoxalase (Manuscript in preparation).
