# 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 · 2020 · $519,833

## Abstract

Project Summary/Abstract
 The advent of the X-ray free electron lasers (XFELs) like the Linac Coherent Light Source (LCLS) at
Stanford has the potential to revolutionize the field of structural studies of biological systems. It is now possible
using the XFELs to determine the structures of enzymes and follow their reactions in real time, at room
temperature. These unprecedented capabilities will open new fields of research, not only in biological sciences
but also in other areas. One bottle-neck in the use of the XFELs has been the lack of a robust method to
introduce the sample into the X-ray interaction region in a continuous manner as the samples are destroyed
after exposure to just one pulse of X-rays, and at the same time minimize the amount of biological sample
used, which are often only available in small quantities. Moreover, it is imperative to be able to trigger the
reactions, by some method, such as using a substrate/chemical compound, or some other stimulus such as
light, or by a temperature jump, or electrical potential so that we can follow the reaction as it happens in real
time, in order to be able to understand how the enzyme functions. This proposal deals with exploring and
constructing robust and versatile sample delivery and reaction triggering methods. Also, when combined with
complementary techniques like spectroscopy and other methods in situ, both global structures and chemical
properties of enzymes can be obtained concurrently, providing insights into the interplay between the protein
structure/dynamics and chemistry at an active site. We will focus on the development of drop-on-demand
methods, mostly based on an acoustic transducer, that will substantially diminish or eliminate any sample
wastage which is a problem with the more commonly used capillary based sample delivery methods that also
have other problems such as clogging that are eliminated with an acoustic droplet ejector. We propose to
develop methods 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. There we can use the X-ray pulses to study the
intermediate states in enzymes that will be generated by substrate (liquid/gas) activation, which covers most of
the enzymatic reactions. Other triggering methods such as light, or temperature jump, or an electric potential
that will be used to study redox active enzyme systems, will be built-in into our sample delivery system.
Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-liquid mixing with micron
size droplet collision methods to achieve faster time resolution that can be followed by subsequent time-
evolution before the X-ray probe.

## Key facts

- **NIH application ID:** 9963327
- **Project number:** 5R01GM126289-04
- **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:** 2020
- **Award amount:** $519,833
- **Award type:** 5
- **Project period:** 2017-09-16 → 2022-06-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9963327

## Citation

> US National Institutes of Health, RePORTER application 9963327, High throughput sample delivery method for time resolved studies of enzyme reactions with X-ray and complementary techniques (5R01GM126289-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9963327. Licensed CC0.

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