Rescue of Misfolded Proteins and Stabilization by Small Molecules - Protein Misfolding and Cellular Stress in Disease and Aging - page 307

1. The protocol here presented is adapted to measure the appar-
ent affinity of ligand compounds to soluble PAH (as concen-
trations for half-maximal binding: C 0.5 ). Prior to measurements,
PAH samples (1 mM subunit) in 20 mM NaHepes, 200 mM
NaCl, pH 7.0, 2.5% DMSO are incubated at room tempera-
ture for 30 min with 0-100 mM compound to allow
equilibration.
2. Intrinsic tryptophan fluorescence spectra are acquired at
25°C, with λ exc = 295 nm (5-nm slit) and emission from 300
to 400 nm (5-nm slit), and corrected for blank emission.
3. Fractional quenching values in the presence of the ligands are
calculated from the decrease in fluorescence at 340 nm at increas-
ing ligand concentrations relative to samples without ligand.
4. C 0.5 for each compound is estimated from fittings to a hyper-
bolic function, including a linear term for inner filter effects:

3.4. Determination
of Binding Affinity
of Positive Hits from
HTP Screening
by Fluorescence
Quenching

(

[ ( ) [ ( )

)

[ ]

F = a ×+ × L F L / C + L

(2)

max 0.5

5. Where F is the experimental fractional quenching, [L] is the
total ligand concentration, F max is the predicted maximal
quenching due to specific ligand binding, and a is a constant
that describes the ligand concentration dependence of the
inner-filter effect.

3.5. Coupled In Vitro
Transcription-
Translation System

1. Coupled in vitro cell free transcription-translation systems
have been very advantageous in previous investigations to
prove the chaperone effect of compounds ( 24, 25 ). In this
in vitro system, compounds can easily be added to the assay,
allowing the synthesis of proteins in the presence of defined
supplementations. The Rapid Translation System (RTS) is
compatible with monitoring protein synthesis in short time
scales in order to follow the kinetics of protein synthesis pre-
vious to saturation (occurring at about 60 min) and analyze
whether the compounds accelerate protein synthesis ( 13 ).
2. In the case of PAH, selective synthesis of active [ 35 S]-labeled
human enzyme is obtained at 37°C by RTS with the enhanced
E. coli lysate (50 ml of the enhanced E. coli lysate), containing
50 mM 35 S-L-Met and the expression vector pcDNA3-WT-
PAH (250 ng) in the absence and presence of hit compounds
(100 mM) in 2.5% DMSO ( 13 ). Two and a half percent
DMSO is also present in control synthesis assays. RTS is car-
ried out at 30°C and 600 rpm.
3. Aliquots (7 ml) are taken from the synthesis assays at increas-
ing times up to 2 h, for example, at 15, 22.5, 30, 60 min, and
2 h. Unlabeled L-Met (6 mM), ribonuclease A (1.4 mg/ml),
and DNase (1.4 mg/ml) are added to stop the synthesis in
each aliquot.