Monday, June 30, 2025 6/30/2025

Structural Studies of Large Dynamic Complexes

Award Number: R35GM131693
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/01/2019
PERIOD OF PERFORMANCE END DATE: 05/31/2024

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Structural Studies of Large Dynamic Complexes - The major overall aim of the research described in this MIRA proposal is to elucidate the structural and dynamic characteristics of large macromolecules and their complexes, which generally contain both well- structured and less-structured dynamic or unfolded regions, using a combination of solid-state methods such as crystallography or cryo-EM and solution methods such as NMR, SAXS and fluorescence. Two major systems have been under study in our lab for several years. Members of the NFκB family of transcription factors are held in the cytoplasm in an inactive state by their bound inhibitors (IκBs) until the cell receives an external signal. NFκB is activated by phosphorylation and ubiquitination of the IκBα, which is then targeted for proteasomal degradation, releasing the NFκB to translocate into the nucleus. In a classical negative feedback mechanism, NFκB upregulates transcription of IκBα in addition to signal-specific stress-response genes: newly-synthesized IκBα kinetically enhances NFκB dissociation from the DNA in a process we have termed molecular stripping. A transient ternary complex intermediate is formed during the stripping process and in an exciting new observation, it was recently shown that the stability of the resting NFκB-IκBα complex in the cytoplasm is enhanced by interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex analogous to the transient NFκB-IκBα-DNA complex formed in the nucleus during molecular stripping. We propose the structural characterization of this NFκB-IκBα-RNA complex using a variety of biophysical techniques, including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray scattering, in collaboration with Dr. John Tainer, and will probe the structural and dynamic differences between the binary and ternary complexes of NFκB, IκBα and DNA, and the ternary NFκB-IκBα-RNA complex using specifically methyl-labeled proteins. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. We propose an innovative method of preparation of a client protein-Hsp90 complex, by reconstituting the chaperone cascade of the eukaryotic cell, but in the context of a cell-free expression system employing bacterial cell extracts. We will prepare the complex of the estrogen receptor ligand-binding domain and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. Our cell-free protocol will include the use of a range of separate bacterial cell extracts containing the over-expressed, folded co-chaperones required according to literature reports for the formation of a stable complex with Hsp90. The advantage of this cell-free system is that it can be tuned to the optimization of complex formation by varying the relative amounts of the component cell extracts.


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: 2025 ( Subtotal = $0 )
2025	2023	SCRIPPS RESEARCH INSTITUTE	10550 N TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	5	11/26/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $0 )
2024	2023	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	5	10/11/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2023 ( Subtotal = $514,710 )
2023	2023	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	5	6/29/2023	NON-COMPETING CONTINUATION	$514,710
														Subtotal = $514,710

Issue Date FY: 2022 ( Subtotal = $514,710 )
2022	2022	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	4	5/12/2022	NON-COMPETING CONTINUATION	$514,710
														Subtotal = $514,710

Issue Date FY: 2021 ( Subtotal = $514,710 )
2021	2021	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	3	5/6/2021	NON-COMPETING CONTINUATION	$514,710
														Subtotal = $514,710

Issue Date FY: 2020 ( Subtotal = $514,710 )
2020	2020	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	2	5/5/2020	NON-COMPETING CONTINUATION	$514,710
														Subtotal = $514,710

Issue Date FY: 2019 ( Subtotal = $514,710 )
2019	2019	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	1	5/28/2019	NEW	$514,710
														Subtotal = $514,710

Grand Total All Awards = $2,573,550

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Structural Studies of Large Dynamic Complexes

Award Number: R35GM131693

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 06/01/2019

PERIOD OF PERFORMANCE END DATE: 05/31/2024

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