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Fig. 8.4
Amino acid changes to KP-10 at the C-terminus and receptor binding. (
a
) KP-10 binds
strongly to GPR54 with an IC
50
of 7.7 nM. (
b
) D -Ser
5
retains binding with an IC
50
of 140 pM
(
c
) Peptide 234 with a D -Ala
1
binds with a similar IC
50
value to KP-10 of 2.8 nM. (
d
) D -Asn at
position 2 and D-Trp at position 3 cannot bind the receptor. Amino acid composition is also shown
for each analogue
Substitutions were then made for the three most N-terminal residues in combination
with Gly
5
and D -Trp
8
. Asn
4
was not substituted in these studies, as little effect was
seen for this residue with alanine screening [
19
,
20
]. Two changes were made at
Tyr
1
; fi rstly, this residue was replaced with D -Tyr
1
(Peptide 230) to test side chain
positioning and then with D -Ala
1
(Peptide 234) to test the importance of side chain
interactions for binding. Peptide 230 was still able to bind to the receptor, but the
displacement of radiolabelled ligand was decreased, whereas peptide 234 could
bind the receptor with similar binding affi nity to KP-10 and exhibited a greater
amount of displacement compared to peptide 230 (Fig.
8.1
). Therefore, we can
assume that Tyr
1
is not very important for binding to the receptor. Two similar
substitutions were made in place of Asn
2
. Peptide 232 incorporated a D -Asn
2
and
peptide 236 had a D -Ala
2
in place of the asparagine removing the charge at this
position. Peptide 232 and peptide 236 could not bind to the receptor (Fig.
8.1
).
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