# High throughput sample delivery method for time resolved studies of enzyme reactions with X-ray and complementary techniques

> **NIH NIH R01** · UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB · 2022 · $608,543

## Abstract

Project Summary/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 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 enzymes 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. 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 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
enzymes of biomedical importance in action, which will contribute to a deeper mechanistic understanding of
these molecular machines, it is essential to synchronize the enzyme in the probed sample volume and initiate
the reaction of interest in a temporally well-defined manner. Methods for reaction initiation can include mixing
with a substrate/chemical compound, or utilize other stimuli such as light, temperature jump, or change in pH or
electrical potential. In the frame of this proposal, we will continue the development of robust and versatile sample
delivery and reaction triggering methods. We will also integrate multi-modal detection methods, combining X-ray
diffraction with complementary in situ spectroscopic techniques to probe both global structures and chemical
properties of enzymes concurrently. We will focus on the development of drop-on-demand methods based on
acoustic transducer technology, but also explore other droplet dispensing technologies and microfluidics to
substantially diminish/eliminate any sample wastage. We will improve the previously developed prototypes for
depositing the drops on a moving support, such as a tape or wheel, that can circulate and is self-cleaning, for
non-stop 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 accommodated...

## Key facts

- **NIH application ID:** 10446972
- **Project number:** 2R01GM126289-05A1
- **Recipient organization:** UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
- **Principal Investigator:** Jan F Kern
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $608,543
- **Award type:** 2
- **Project period:** 2017-09-16 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10446972

## Citation

> US National Institutes of Health, RePORTER application 10446972, High throughput sample delivery method for time resolved studies of enzyme reactions with X-ray and complementary techniques (2R01GM126289-05A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10446972. Licensed CC0.

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