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.
