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

Award Number: R01GM126289
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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 by modeling approaches to design chemical mixing experiments and use feedback from measurements to optimize the design. These will be implemented at SR and XFEL beam lines and made available for the broader structural enzymology user community.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = $588,716 )
2024	2024	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	7	5/8/2024	NON-COMPETING CONTINUATION	$588,716
														Subtotal = $588,716

Issue Date FY: 2023 ( Subtotal = $614,074 )
2023	2023	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	002	6	6/28/2023	NON-COMPETING CONTINUATION	$614,074
2023	2022	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	001	5	6/8/2023	COMPETING CONTINUATION	$15,323
2023	2020	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	4	6/8/2023	NON-COMPETING CONTINUATION	-$15,323
														Subtotal = $614,074

Issue Date FY: 2022 ( Subtotal = $608,543 )
2022	2022	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	001	5	6/14/2022	COMPETING CONTINUATION	$608,543
2022	2020	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	4	6/6/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $608,543

Issue Date FY: 2020 ( Subtotal = $519,833 )
2020	2020	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	4	6/3/2020	NON-COMPETING CONTINUATION	$519,833
														Subtotal = $519,833

Issue Date FY: 2019 ( Subtotal = $642,044 )
2019	2019	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	3	6/24/2019	NON-COMPETING CONTINUATION	$545,422
2019	2019	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	001	3	9/17/2019	SUPPLEMENT FOR EXPANSION	$96,622
														Subtotal = $642,044

Issue Date FY: 2018 ( Subtotal = $547,037 )
2018	2018	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	2	6/25/2018	NON-COMPETING CONTINUATION	$547,037
														Subtotal = $547,037

Issue Date FY: 2017 ( Subtotal = $548,914 )
2017	2017	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	1	9/16/2017	NEW	$548,914
														Subtotal = $548,914

Grand Total All Awards = $4,069,161

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

Award Number: R01GM126289

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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