Enabling time resolved studies of small molecule-protein interactions with pharmacological relevance using serial crystallography

The National Institute of General Medical Sciences awarded $1,324,670 to the Regents of the University of California (operating Lawrence Berkeley National Laboratory) on July 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to enable time-resolved studies of small molecule-protein interactions with pharmacological relevance using serial crystallography.

The research develops methods to follow structural changes in proteins in real time and under near-physiological conditions at room temperature by leveraging X-ray free electron lasers (XFELs) such as the Linac Coherent Light Source at Stanford and time-resolved crystallography capabilities at synchrotron radiation sources. The work aims to better understand reaction mechanisms and binding behavior in protein-ligand systems of pharmacological importance. A core technical challenge the project addresses is developing robust, continuous sample-introduction methods that minimize required sample amounts, since the "probe before destroy" approach used in XFEL experiments requires sample replacement after each X-ray exposure and biological samples are often available in limited quantities. The project also emphasizes developing fast, efficient, and homogeneous mixing means to obtain molecular-level dynamic data of proteins interacting with small molecules.

Performance takes place in Berkeley, California. The period of performance runs from July 1, 2026, through June 30, 2031.

Abstract

One of the new frontiers in structural enzymology is the expansion from a three-dimensional to a truly four-dimensional approach by adding the time dimension to structural studies. While conventional synchrotron radiation (SR) crystallography and cryo electron microscopy allow the determination of structures in minute detail they are in most cases performed on frozen static samples. With the advent of X-RAY free electron lasers (XFELS) like the linac coherent light source (LCLS) at stanford, and the development of the "probe before destroy" concept IT now is possible to follow structural changes in proteins in real time and under close to physiological conditions at room temperature (RT). Driven by the success of XFELS and recent advances in detector technology and storage ring and beam line design, several SR sources are also starting to offer time resolved crystallography at RT. These unprecedented capabilities will open new fields of research, not only in biomedical sciences but also in many other areas. We are aiming to utilize these recent advances to study a number of proteins and their interaction with small molecules of pharmacological importance with the goal of better understanding reaction mechanisms and binding behavior in these systems. Due to the "probe before destroy approach" utilized here, the samples generally need to be replaced after a single X-RAY exposure. As biological samples of interest (proteins as well as ligands/inhibitors) are often only available in scarce amounts, IT is mandatory to develop a robust method to introduce the sample into the X-RAY interaction region in a continuous manner that minimizes the required sample amount. In order to obtain true "molecular movies" of proteins interacting with small molecules, IT is essential to develop means of fast, efficient and homogeneous mixing of the protein crystalline sample with the small molecule. In the frame of this proposal, we will developmen robust and versatile sample delivery and mixing/ triggering methods. We will improve the previously developed prototypes for depositing the drops on a moving support, for non-stop automated continuous operation at the xfel or SR facility. Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-gas and liquid-liquid mixing, including with micron size droplet collision methods to achieve faster time resolution. Experiments on well-defined enzyme model systems will be used first to benchmark the methods, followed by measurements on cytochrome P450 variants that are essential for survival of specific pathogens, oxygen sensing proteins that are involved in the cycling between dormant and virulent forms of tuberculosis, but also mono-FE and DI-FE containing systems involved in oxygen sensing in humans, natural antibiotics synthesis in fungi and FE uptake in mycoplasma pneumonia and beta lactamases involved in antibiotic resistance.