Rescue of Misfolded Proteins and Stabilization by Small Molecules - Protein Misfolding and Cellular Stress in Disease and Aging - page 301

and/or degradation can be measured using in vitro transcription-
translation systems ( 17 ). The protocols have been adapted for the
search of pharmacological chaperones for a soluble protein: phe-
nylalanine hydroxylase (PAH), and a membrane one: a G protein-
coupled receptor (GPCR).
Over the last years, it has been shown that misfolding of
mutated PAH - a cytosolic protein involved in hepatic catabolism
of L-Phe - is the molecular basis of the genetic metabolic disease
phenylketonuria (PKU; OMIM 261600) ( 18 ). Recent efforts
have concentrated in the search of stabilizing compounds with
therapeutic potential as pharmacological chaperones to treat PKU
( 13 ). On the other hand, GPCRs represent a large family of inte-
gral membrane proteins involved in signal transduction, and pro-
vide a very important target for therapeutic discovery ( 19 ).
Microscale screening assays for selection of stabilizing ligands of
GPCRs have also been recently developed ( 7 ).

2. Materials

2.1. HTP Screening
of Stabilizing Ligands
for Soluble Proteins
by the Fluorescence
Thermal Shift Assay

1. Fluorescence dyes such as 8-anilino 1-naphthalene sulfonic
acid (ANS) or Sypro Orange (Molecular Probes, see Note 1).
The fluorescence properties of these dyes are environment-
sensitive; they are essentially non-fluorescent in water and
become substantially fluorescent when bound to membranes
or hydrophobic regions of proteins, thus being valuable indi-
cators of protein folding.
2. FluoDia T70 High Temperature Fluorescence Microplate
Reader (PTI). It can be substituted by an iCycler iQ Real
Time PCR Detection System (Bio-Rad).
3. 96-well microplates (ThermoFast 96 skirted from Thermo
Scientific or 96-well thin-wall PCR plates if the iCycler iQ is
used).
4. Compound diversity collection (e.g., from the Maybridge
HitFinder Collection database, Maybridge Ltd., or the
MyriaScreen Diversity Collection, Sigma-Aldrich). The com-
pounds, which are originally dissolved in 100% dimethyl sul-
foxide (DMSO), are added at a final concentration of 100 mM
and 2.5% DMSO to the 96-well microplates containing the
protein solutions.
5. Protein sample to be analyzed, e.g., recombinant human
PAH, wild-type or mutants associated with PKU, expressed in
Escherichia coli and purified as described ( 20 ). The protein
samples are prepared as 5-10 mg/ml in 20 mM Hepes-
NaOH, pH 7.0, 200 mM NaCl, and added at a final concen-
tration in the plates of 0.1 mg/ml PAH (~2 mM PAH
subunit).