Characterizing Enzyme Reactions in Microcrystals for Effective Mix-and-Inject Experiments using X-ray Free-Electron Lasers

Abstract

Mix-and-inject serial crystallography is an emerging technique that utilizes X-ray free-electron lasers (XFELs) and microcrystalline samples to capture atomically detailed snapshots of biomolecules as they function. Early experiments have yielded exciting results; however, there are limited options to characterize reactions in crystallo in advance of the beamtime. Complementary measurements are needed to identify the best conditions and timescales for observing structural intermediates. Here, we describe the interface of XFEL compatible mixing injectors with rapid freeze-quenching and X-band EPR spectroscopy, permitting characterization of reactions in crystals under the same conditions as an XFEL experiment. We demonstrate this technology by tracking the reaction of azide with microcrystalline myoglobin, using only a fraction of the sample required for a mix-and-inject experiment. This spectroscopic method enables optimization of sample and mixer conditions to maximize the populations of intermediate states, eliminating the guesswork of current mix-and-inject experiments.

Figure 1.

Schematic of a MISC experiment and mixing injector. Microcrystals and an activating ligand are combined in the mixer and subsequently propelled into the X-ray beam by the GDVN. Snapshot diffraction images are collected by the detector.

Figure 2.

Cartoon of the RFQ process developed for mixing injectors. (a) Reacting sample is quenched in cryogenic isopentane. (b) Quenched sample is transferred to the cold flow packing system. (c) Flow packing system is pressurized by cold nitrogen gas. (d) Sample accumulates behind the filter as isopentane is drained.

Figure 3.

EPR data for the myoglobin and 15 mM azide reaction in the solution state. EPR intensities are provided in relative units. For high field strengths, EPR intensities are multiplied by 5 for ease of viewing. (a) EPR spectra of the high- and low-spin solution standards. Peak-to-peak intensities used in data analysis are illustrated. (b) Time-resolved EPR spectra of myoglobin/azide reaction. Curves are normalized by the total myoglobin concentration (see the Supporting Information and Figure S5 for details). (c) Percent of the initial state (F_HS(t)) remaining as a function of time.

Figure 4.

EPR data for the reaction between myoglobin microcrystals and 114 mM azide. EPR intensities are shown in relative units. (a) EPR spectra of the high- and low-spin crystal standards. (b) Time-resolved EPR spectra of the myoglobin/azide reaction normalized for myoglobin concentration (see the Supporting Information). The spectra for timepoints with duplicate measurements are averaged. (c) Percent of the initial state remaining as a function of time for the reaction in microcrystals (circles) and solution at the same azide concentration for comparison (black triangle). The 2× label indicates two overlapping datapoints at 250 ms.