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Single-molecule Measurements of Membrane-protein Folding and Ligand-Interaction Energetics in Bacteriorhodopsin and the Diabetes-insipidus-involved Vasopressin Receptor 2

Award Number: R00GM140439
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: 03/01/2021
PERIOD OF PERFORMANCE END DATE: 08/31/2026

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Single-molecule Measurements of Membrane-protein Folding and Ligand-Interaction Energetics in Bacteriorhodopsin and the Diabetes-insipidus-involved Vasopressin Receptor 2 - Project Summary/Abstract Human arginine vasopressin receptor 2 (AVPR2) is an -helical membrane protein expressed in the collecting ducts of the kidneys involved in regulating urine volume. Mutations of AVPR2 at some 96 sites are known to cause nephrogenic diabetes insipidus (NDI), likely by promoting misfolding. The vasopressin antagonist drugs (“vaptans”) have been shown to rescue cell-surface expression, putatively by acting as chaperones stabilizing the native folded state. Thus, understanding the relative energetics of the folded and misfolded states in the presence and absence of ligands would shed light on the native structural dynamics of AVPR2 and how those dynamics are changed by disease-causing mutations. Such results would be relevant to NDI, and also more generally to diseases arising from G-protein coupled receptors (GPCRs). However, the established biochemical technique of chemical denaturation in detergent micelles that is used to measure membrane-protein thermodynamic stability (G) and its change upon mutation or ligand binding (G) suffers from several limitations that make it unsuitable for studies of AVPR2. In particular, the non-native detergent environment, the poorly defined denatured state with significant residual secondary structure, and the need to extrapolate from high denaturant concentration cause the measured energetics to poorly reflect the underlying, biologically relevant molecular values. Most significantly, chemical-denaturation-based techniques have never been successfully applied to GPCRs because GPCRs do not globally refold when the denaturant is removed. These shortcomings motivate the overall aim of this proposal: to develop alternate techniques for measuring membrane-protein energetics, based on force-induced unfolding rather than chemical denaturation. Such techniques, implemented on an atomic force microscope (AFM), can study membrane proteins in the native lipid bilayer and obviate the problem of globally reversible unfolding by probing a small portion of the protein at a time. Work during the postdoctoral K99 phase will use the model membrane protein bacteriorhodopsin (bR) to further develop these force-based techniques. Two particular aims will be achieved: (1) measurement of point-mutant free energy changes of bR in its native bilayer and without confounding chemical denaturant and (2) quantification of the energetics of a photo-activated ligand isomerization in bR. Completing this work during the K99 phase will establish the basis for the aim of the independent R00 phase: to elucidate the folding and ligand-interaction energetics of AVPR2 using these new techniques. In addition to providing specific insight into AVPR2 folding and misfolding, this work will establish a new paradigm in which energetic measurements can be made directly in biomedically relevant systems like AVPR2, rather than just in model systems. The transition to independence will also be facilitated by training during the K99 phase, most notably in the expression and purification of GPCR samples. The University of Colorado provides world-class facilities for carrying out this work, and co-mentors will offer expertise in both single-molecule AFM experiments and membrane-protein biochemistry.


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 = $249,000 )
2025	2025	CLEMSON UNIVERSITY	201 SIKES HALL	CLEMSON	SC	29634	PICKENS	USA	Biomedical Research and Research Training	000	5	7/30/2025	NON-COMPETING CONTINUATION	$249,000
														Subtotal = $249,000

Issue Date FY: 2024 ( Subtotal = $273,102 )
2024	2024	CLEMSON UNIVERSITY	201 SIKES HALL	CLEMSON	SC	29634	PICKENS	USA	Biomedical Research and Research Training	001	4	8/27/2024	NON-COMPETING CONTINUATION	$249,000
2024	2023	CLEMSON UNIVERSITY	201 SIKES HALL	CLEMSON	SC	29634	PICKENS	USA	Biomedical Research and Research Training	000	3	1/9/2024	EXTENSION WITH OR WITHOUT FUNDS	$24,102
														Subtotal = $273,102

Issue Date FY: 2023 ( Subtotal = $249,000 )
2023	2023	CLEMSON UNIVERSITY	201 SIKES HALL	CLEMSON	SC	29634	PICKENS	USA	Biomedical Research and Research Training	000	3	8/15/2023	EXTENSION WITH OR WITHOUT FUNDS	$249,000
														Subtotal = $249,000

Grand Total All Awards = $771,102

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Single-molecule Measurements of Membrane-protein Folding and Ligand-Interaction Energetics in Bacteriorhodopsin and the Diabetes-insipidus-involved Vasopressin Receptor 2

Award Number: R00GM140439

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: 03/01/2021

PERIOD OF PERFORMANCE END DATE: 08/31/2026

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