Rescue of Misfolded Proteins and Stabilization by Small Molecules - Protein Misfolding and Cellular Stress in Disease and Aging - page 306

which the extent of covalent coupling of receptor cysteines to
the thiol-reactive dye CPM is monitored as a function of time
at constant temperature ( 16 ).
2. Thermal stability data at 35°C are collected in the presence of
CPM and of 1 M GnHCl (other concentrations of GnHCl
may be evaluated).
3. The protein is prepared in a concentration of about 0.5-1 mg/
ml in 50 mM Na-Hepes, pH 7.5, 150 mM NaCl, containing
2 mg of CPM dye (diluted from the 4 mg/ml stock in dim-
ethyl formamide) and incubated at 4°C for 10 min.
4. Various combinations of ligands are then added to the mix-
ture at 2× final concentration and allowed to equilibrate for
30 min at 4°C before the final dilution to working volume
with the appropriate concentration of GnHCl in the assay
buffer. Final volume of the assay is 60 ml.
5. Data are collected as soon as possible after the addition of the
chemical denaturant to reduce the degree of unfolding prior
to measurement.
6. Fluorescence of the CPM dye is measured every minute for
3 h on a fluorescent plate reader using 340-nm excitation fil-
ter with a 35-nm bandpass and a 475-nm emission filter with
a 20-nm bandpass.
7. Data are fitted to a single exponential decay curve using
GraphPad Prism Software so that first-order rate constants
( k ) for denaturation and half-lives ( t 1/2 ) of protein denatur-
ation ( t 1/2 =−ln(0.5)/ k ) are obtained (see Fig. 3 ). The assay
aims to select compounds that increase t 1/2 more than two-
fold relative to the ligand-free protein.
Fig. 3. Isothermal denaturation assay to determine ligand-induced stabilization of membrane proteins. Isothermal CPM
determination of the half-life of denaturation of a recombinant form of human b 2 -adrenergic receptor ( b 2 AR) in the pres-
ence of 1 M GnHCl at 35°C, with and without both cholesterol (CHS) and timolol (Tim). The thickness of the line represents
the 95% confidence interval over three replicates, and the fitted half lives are indicated next to the respective curves.
Both TIM and CHS cause an approximate fivefold increase in half-life under these conditions. In combination, the effect
is almost 16-fold relative to the ligand-free apo protein. Copied with permission from ( 16 ).