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0:00
As I stated in the
introduction to
Part 1 of this talk on PSD-95,
my name is Johannes Hell.
I am a Professor
of Pharmacology at
the University of
California, Davis,
Vice-Chair for
Academic Development
and the Director of our T-32
training program
in Pharmacology.
0:21
This second part will
delineate our work on
the structural details
that mediate binding of
the PDZ domains of PSD-95
to target recognition
sites on binding partners,
like the NMDAR receptor
GluN2 subunits
or the AMPAR receptor
auxiliary TARP subunits.
0:42
Starting points for
our modeling were
binding assays with
each peptide measuring
fluorescence polarization
upon addition
of increasing amounts of
individual purified PDZ domains,
which we expressed as GST
fusion proteins in E.coli.
When fluorescent molecules are
excited with polarized light,
they will maintain the
vector of the wave
when emitting fluorescence,
but tumbling will degrade the
orientation of this vector.
Small molecules tumble
faster than large ones.
When PDZ domains bind
to our peptides,
the tumbling rate will decrease
and polarization will
be better preserved.
Thus, the increase
in polarization
will reflect the binding of
PDZ domains to peptides.
We determined the K_D values
of a number of PDZ domains,
which is given as half-maximal
binding concentration
of such titration curves,
as shown here for PDZ1 and
as shown here for PDZ1
and PDZ2 from PSD-95,
and SAP102 against the peptide
visible type sequence of GluN2B.
These experiments show
that PDZ2 domains
have a significantly higher
affinity than PDZ1 domains
and that SAP102 has
a higher affinity
than PSD-95 for GluN2 subunits.
