Rescue of Misfolded Proteins and Stabilization by Small Molecules - Protein Misfolding and Cellular Stress in Disease and Aging - page 302

2.2. HTP Screening
of Stabilizing Ligands
for Membrane Proteins
by the Fluorescence
Thermal Shift Assay

1. Thiol-reactive N -[4-(7-diethylamino-4-methyl-3-coumarinyl)
phenyl]maleimide (CPM) from Invitrogen (see Note 2). Prior
to use the dye stock is diluted in dye dilution buffer (20 mM
Hepes-NaOH, pH 7.5, 200 mM NaCl, 0.025% n -dodecyl-b-
d -maltoside (DDM) (Sigma), incubated for 5 min at room
temperature, and used immediately while protected from light
to reduce photobleaching. Optimal dilution of the CPM dye
should be determined empirically for each tested protein, but
a 1:40 dilution is appropriate in most cases.
2. FluoDia T70 High Temperature Fluorescence Microplate
Reader (PTI, Birmingham, NJ).
3. 96- or 384-well microplates.
4. Compound diversity collection (see 2.1, above).
5. Protein to be analyzed, for example, recombinant human b 2 -
adrenergic receptor (b 2 AR), a well-characterized GPCR,
expressed and purified from baculovirus-infected Sf9 cells as
described ( 21 ).

2.3. Isothermal
Denaturation Assay
to Determine Ligand-
Induced Stabilization
of Membrane Proteins

1. CPM prepared as a 4 mg/ml stock in dimethyl formamide.
2. 1 M guanidinium hydrochloride (GnHCl). This chemical
denaturant is included to speed the process of unfolding,
thereby minimizing the effects of dehydration on data pro-
cessing errors.
3. Membrane protein, for example, b 2 AR.
4. 96- or 384-well microplates.
5. A Genios Pro fluorescent plate reader (Tecan Systems, Inc.,
San Jose, CA).
6. GraphPad Prism program (GraphPadPrism v.4.00 for Windows,
Graphpad Sofware, San Diego, CA).

2.4. Determination
of Binding Affinity
of Positive Hits from
HTP Screening
by Fluorescence
Quenching
2.5. Coupled In Vitro
Transcription-
Translation System

1. PAH (1 mM subunit) in 20 mM Hepes-NaOH, pH 7.0,
200 mM NaCl.
2. 0-100 mM of the hit compounds in 2.5% DMSO.
3. Spectrofluorimeter, for example, Cary Eclipse spectrofluo-
rimeter (Varian Inc., Mulgrave, Victoria, Australia).

1. Rapid Translation System (RTS) (RTS ProteoMaster 500;
Roche Molecular Biochemicals, Roche Diagnostics GmbH,
Mannheim, Germany).
2. Enhanced E. coli lysate kit for RTS (RTS 100 E. coli HY Kit,
Roche Molecular Biochemicals).
3. 35 S-L-Met (Amersham Pharmacia Biotech, Piscataway, NJ).
4. Expression vector with T7 promotor including the gene of
the protein to be expressed (e.g., pcDNA3-wt-PAH).
5. Hit compounds at defined concentrations.