Thursday, May 2, 2024 5/2/2024

Development of biophysical approaches to investigate high-resolution structure and dynamics of membrane proteins

Award Number: R35GM139573
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Abstract Functional reconstitution of membrane proteins has been the major roadblock for the application of NMR and other biophysical techniques to investigate their high-resolution dynamic structures in a native membrane environment. In this application, we propose to develop approaches to enable high-resolution structural studies of membrane proteins and protein-protein complexes by a variety of biophysical techniques. We will develop nanodisc technology for detergent-free direct extraction and functional reconstitution of membrane proteins for structural studies of a variety of membrane proteins including single-pass transmembrane proteins (such as mammalian cytochromes and heme oxygenase) and integral membrane proteins (including GPCRs and Guanidine exporter). Synthetic polymers developed in our laboratory exhibit the ability to form nanodiscs with easily controllable sizes (from ~8 to ~60 nm diameter), are stable against pH and divalent metal ions and capable of directly extracting membrane proteins. Our preliminary results demonstrate that these nanodiscs (<20 nm diameter) and macro-nanodiscs (>20 nm diameter) represent an exciting system for solution and solid-state NMR studies of membrane proteins. We also propose to use the newly developed nanodisc technology and NMR approaches to investigate the structural interactions of mammalian cytochrome-P450 (P450) with its redox partners (P450-reductase (CPR) and cytochrome-b5 (b5)) to better understand how redox partners regulate P450 catalysis and how P450s metabolize chemically diverse substrates. The structural aspects pertaining to the catalytic activity of P450s continue to remain elusive due to a lack of high-resolution structures in their full-length, active forms. Presently, structural studies of P450s are restricted to various truncated mammalian and water-soluble bacterial P450 homologs. In this study, we will investigate the structure, dynamics and transmembrane domain orientation of full-length mammalian P450s (2B4, 3A4 and 3A5 isoforms) alone and in complex with its redox partner b5 and CPR, incorporated in nanodiscs, using a combination of high-resolution solution and solid-state NMR techniques. We will also investigate the ternary P450-b5-CPR complex in nanodiscs in the presence of substrates to elucidate the molecular origin of the strikingly different effects CPR and b5 have on P450 2B4 catalysis. The outcome of the proposed studies on P450-redox complexes will provide structure and dynamics/function principles regulating P450 metabolism of a wide variety of substrates. The results obtained from this study will also be useful to design potent drugs to ultimately treat and prevent diseases including cancer.


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 = $359,909 )
2024	2024	FLORIDA STATE UNIVERSITY	874 TRADITIONS WAY	TALLAHASSEE	FL	32306	LEON	USA	Biomedical Research and Research Training	002	4	1/30/2024	NON-COMPETING CONTINUATION	$356,625
2024	2022	FLORIDA STATE UNIVERSITY	874 TRADITIONS WAY	TALLAHASSEE	FL	32306	LEON	USA	Biomedical Research and Research Training	001	3	12/13/2023	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$208,221
2024	2022	REGENTS OF THE UNIVERSITY OF MICHIGAN	1109 GEDDES AVE, SUITE 3300	ANN ARBOR	MI	48109	WASHTENAW	USA	Biomedical Research and Research Training	000	2	12/13/2023	NON-COMPETING CONTINUATION	-$204,937
														Subtotal = $359,909

Issue Date FY: 2023 ( Subtotal = $0 )
2023	2022	REGENTS OF THE UNIVERSITY OF MICHIGAN	503 THOMPSON ST	ANN ARBOR	MI	48109	WASHTENAW	USA	Biomedical Research and Research Training	000	2	12/2/2022	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2022 ( Subtotal = $386,143 )
2022	2022	REGENTS OF THE UNIVERSITY OF MICHIGAN	503 THOMPSON ST	ANN ARBOR	MI	48109	WASHTENAW	USA	Biomedical Research and Research Training	001	2	6/30/2022	NON-COMPETING CONTINUATION	$38,615
2022	2022	REGENTS OF THE UNIVERSITY OF MICHIGAN	503 THOMPSON ST	ANN ARBOR	MI	48109	WASHTENAW	USA	Biomedical Research and Research Training	000	2	11/22/2021	NON-COMPETING CONTINUATION	$347,528
														Subtotal = $386,143

Issue Date FY: 2021 ( Subtotal = $386,143 )
2021	2021	REGENTS OF THE UNIVERSITY OF MICHIGAN	503 THOMPSON ST	ANN ARBOR	MI	48109	WASHTENAW	USA	Biomedical Research and Research Training	000	1	12/23/2020	NEW	$386,143
														Subtotal = $386,143

Grand Total All Awards = $1,132,195

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Development of biophysical approaches to investigate high-resolution structure and dynamics of membrane proteins

Award Number: R35GM139573

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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