Chaotic advection mixer for capturing transient states of diverse biological macromolecular systems with time-resolved small-angle X-ray scattering

Abstract

Time-resolved mixing experiments, in which a microfluidic device is used to rapidly mix two species together to initiate a reaction, are a powerful tool to collect snapshots of the time-progression of macromolecular interactions. These mixing experiments are compatible with a variety of experimental techniques as structural probes, but Small Angle X-ray Scattering (SAXS) is particularly well suited to these studies as it can capture changes in the overall shape, size, and level of compactness of biological macromolecules with unconstrained motions in solution. Flowfocused diffusive mixers have been used successfully for time-resolved SAXS experiments, but they require that one of the reactants is small and highly soluble to achieve the rapid diffusion required to uniformly initiate a reaction. Although this requirement can be easily met for a broad range of biological macromolecule-ligand systems, many drug targets tend to be partially hydrophobic, not highly soluble, and not easily available in large quantities. Additionally, diffusive mixers preclude the study of the interaction of two large biological macromolecules, such as two proteins or a protein-nucleic acid system, as the diffusion times for these larger molecules can be longer than the reaction times of interest.We present a novel coupling of the Kenics-style chaotic advection mixer with SAXS to study diverse classes of macromolecular interactions, including reactions between two large biological macromolecules or one large biological macromolecule and a ligand. The mixer is comprised of a series of eight helical elements with alternating left-and right-handedness. Rapid mixing is achieved by creating ultra-thin layers of each species via baker's transformations (stretching, splitting, and layering of the liquids), so even large proteins can be mixed in as fast as a few milliseconds. The mixer itself was fabricated with a NanoScribe 3D Printer and our sample cell design presents a sufficiently large observation region which permits a good signal to noise ratio. Timepoints from 10-2000 ms can be reached by changing flowrates or the position of the X-ray beam relative to end of the Kenics mixer. We used this mixer to study a variety of different biological questions, such as RNA folding, protein conformational changes, protein-protein associations, and protein-nucleic acid complex formation. With this mixer, we captured transient reaction states that evade observation by typical equilibrium measurements and visualization of these short-lived states can elucidate the mechanism of these reactions or reveal the initial stages of comple

Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death

“…To gain a better mechanistic understanding of how LM11 and Ca 2+ induce TG2 to adopt an irreversible open conformation, we used time-resolved SAXS (TR-SAXS). A 3D printed Kenics-style chaotic advection mixer (66) was used to rapidly combine two fluids for reaction initiation (Fig. 6A), with the flow rates and position of the X-ray beam adjusted to facilitate timepoints ranging from 32 – 631 ms.…”

“…6B, Tables S4 and S5). The first time series (32, 63, 100, 316, and 631 ms) was obtained by mixing TG2 with Ca 2+ alone (originally published in (66), SASDRE3-SASDRP3). In the absence of LM11, Ca 2+ binding induces a slow conformational change for approximately 100 ms with an Rg of 40.7±0.4 Å (Fig.…”

“…Time-resolved SAXS measurements were taken as described in our previous work (Zielinski et al, 2023). Briefly, a Kenics-style chaotic advection mixer was used to rapidly combine two species just prior to data collection.…”

“…Additionally, SAXS can be combined with time-resolved techniques to study the dynamics of such structural transitions. In particular, stop-flow (62), diffusive (63), turbulent (64), and chaotic advection (65, 66) mixers have all been successfully coupled to SAXS. These approaches rapidly mix two molecules together and then capture snapshots at different timepoints to understand the mechanism of a reaction.…”