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NEW CHEMICAL PROBES ENABLE MASS SPECTROMETRY-BASED FOOTPRINTING OF HUMAN PROTEIN STRUCTURE IN LIPID MEMBRANES AND CELLS

Award Number: R01GM131008
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 Mass spectrometry (MS) based footprinting is emerging as a powerful means to answer biological questions about membrane proteins (MPs), which participate in almost all physiological processes and represent more than 60% of drug targets. This approach affords sufficient structural information for the dynamic, native conformations and interactions of MPs in cells, which are beyond the reach of traditional structural methods (e.g., cryo-EM and crystallography). This bottom-up MS footprinting is complementary to but potentially more informative than top-down native MS, which does not provide spatial resolution for MPs and is conducted in the nonnative gas phase. Here we propose to continue development of novel MS footprinting methods in live cells and native membranes. Our objective is to design, prepare, test, and improve chemical probes that provide high footprinting coverage. We will then apply them to reveal drug interactions and cellular trafficking regulation of a glucose transporter, GLUT1, a prominent anticancer drug target and a model MP representing ~ 25% of known transport proteins. MS footprinting of MPs, however, poses three major challenges. 1) MPs, which are hydrophobic and buried in lipid bilayers, are resistant to traditional probes (e.g., HDX, •OH radicals) that penetrate poorly and give insufficient labeling. 2) Aliphatic side chains of transmembrane regions contain C–H and C-C bonds that are unreactive with most chemical probes. 3) The footprinting needs to be conducted in cells or membranes to maintain native conformation and interaction of MPs. Our hypotheses are: (1) Complementary modifications of C-H and X–H bonds by free radicals produced photochemically and by nucleophilic reagents maximize footprinting coverage. (2) Tuning the hydrophobicity of the reagents or their precursors allows access to membrane-embedded MPs. (3) Novel membrane fusion techniques introduce inert footprinters into live cells and native membranes for subsequent photoactivated footprinting. Our hypotheses are built on extensive preliminary data. Three years of funding supported publication of 18 papers in high-profile journals. A significant example describes laser activation of TiO2 nanoparticles attached to liposomes to generate high local concentrations of radicals. Simultaneous membrane poration permits radical entry to footprint with sufficient structural resolution that reports the ligand-binding sites and rocker-switch motions of GLUT1. Building on these successes, we will pursue two specific aims: (1) develop new chemical probes for MS footprinting of MPs; and (2) conduct comprehensive footprinting in native membranes and live cells to reveal anticancer drug interactions and trafficking regulations of GLUT1. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish bio-orthogonal footprinters that afford comprehensive coverage of both hydrophobic and hydrophilic regions of MPs and reveal drug interactions and structural regulation of human MPs under inarguable native settings. The impact of the proposed approach should readily expand because MS-based footprinting can be broadly applied in structural proteomics to expedite drug discovery and structural studies of cellular processes.


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 = $463,905 )
2024	2024	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63110	SAINT LOUIS CITY	USA	Biomedical Research and Research Training	002	6	2/13/2024	NON-COMPETING CONTINUATION	$417,514
2024	2024	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63110	SAINT LOUIS CITY	USA	Biomedical Research and Research Training	003	6	6/17/2024	NON-COMPETING CONTINUATION	$46,391
2024	2023	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63110	SAINT LOUIS CITY	USA	Biomedical Research and Research Training	001	5	11/1/2023	COMPETING CONTINUATION	$39,954
2024	2022	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63110	SAINT LOUIS CITY	USA	Biomedical Research and Research Training	000	4	11/1/2023	NON-COMPETING CONTINUATION	-$39,954
														Subtotal = $463,905

Issue Date FY: 2023 ( Subtotal = $465,693 )
2023	2023	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	5	4/20/2023	COMPETING CONTINUATION	$465,693
2023	2022	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	001	4	4/21/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $465,693

Issue Date FY: 2022 ( Subtotal = $409,750 )
2022	2022	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	001	4	6/27/2022	NON-COMPETING CONTINUATION	$40,977
2022	2022	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	4	2/9/2022	NON-COMPETING CONTINUATION	$368,773
														Subtotal = $409,750

Issue Date FY: 2021 ( Subtotal = $659,750 )
2021	2021	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	001	3	8/23/2021	SUPPLEMENT FOR EXPANSION	$250,000
2021	2021	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	3	2/12/2021	NON-COMPETING CONTINUATION	$409,750
														Subtotal = $659,750

Issue Date FY: 2020 ( Subtotal = $409,316 )
2020	2020	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	2	2/17/2020	NON-COMPETING CONTINUATION	$409,316
														Subtotal = $409,316

Issue Date FY: 2019 ( Subtotal = $407,149 )
2019	2019	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	1	1/30/2019	NEW	$407,149
														Subtotal = $407,149

Grand Total All Awards = $2,815,563

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NEW CHEMICAL PROBES ENABLE MASS SPECTROMETRY-BASED FOOTPRINTING OF HUMAN PROTEIN STRUCTURE IN LIPID MEMBRANES AND CELLS

Award Number: R01GM131008

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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