Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.
Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.
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-andinject 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.
Structure-based drug design (SBDD) is a prominent method in rational drug development and has traditionally benefitted from the atomic models of protein targets obtained using X-ray crystallography at cryogenic temperatures. In this perspective, we highlight recent advances in the development of structural techniques that are capable of probing dynamic information about protein targets. First, we discuss advances in the field of X-ray crystallography including serial room-temperature crystallography as a method for obtaining high-resolution conformational dynamics of protein-inhibitor complexes. Next, we look at cryogenic electron microscopy (cryoEM), another high-resolution technique that has recently been used to study proteins and protein complexes that are too difficult to crystallize. Finally, we present small-angle X-ray scattering (SAXS) as a potential high-throughput screening tool to identify inhibitors that target protein complexes and protein oligomerization.
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
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