Università degli Studi di MilanoFacoltà di Farmacia
Modelling the folding of transmembrane proteins using a novel fragmental
approach: the human ghrelin receptor and the glutamate transporter EAAT1
Alessandro Pedretti & Giulio Vistoli
The fragmental approach1
Aminoacid sequenceof the transmembrane protein
Aminoacid sequenceof the transmembrane protein
Folding prediction of each fragment
Folding prediction of each fragment
Assembling using a global template
Assembling using a global template
Fragmentation in structural domains
Fragmentation in structural domains
Global template
The fragmental approach2
Side chains buildingSide chains building
Rough modelRough model
Final modelFinal model
MM/MD refinementMM/MD refinement
Molecular dockingMolecular docking
Ligand-protein complexesLigand-protein complexes
LignadsLignads
Model validation
HGHS-R1a ligand binding sites
TM6
TM3
Arg 283
Glu 124
N
NH
O
NH
OH
O
NH3
+
Pro 192 Pro 200
Ala 204 Val 2
05
Phe 119 Arg 199
Glu 124
Ser 123
Asn 188Thr 190 Tyr 106
Arg 107
Tyr 106Arg 107Trp 193
N
NH
O
NH
OH
O
NH3
+
Pro 192 Pro 200
Ala 204 Val 2
05
Phe 119 Arg 199
Glu 124
Ser 123
Asn 188Thr 190 Tyr 106
Arg 107
Tyr 106Arg 107Trp 193
N
NH
O
NH
OH
O
NH3
+
Arg 283 Phe 222Phe 226
Phe 220Tyr 284Phe 286
Ser 207Gln 299
Val 182Ile 219Ile 300
N
NH
O
NH
OH
O
NH3
+
Arg 283 Phe 222Phe 226
Phe 220Tyr 284Phe 286
Ser 207Gln 299
Val 182Ile 219Ile 300
Spiroindane derivativeEC50 = 0.6 nM
Polar
Apolar
QSAR analysis
pEC50 = 6.06 (0.35) – 0.070 (0.01) x Scorepol n = 35; r2 = 0.57; q2 = 0.51 s = 0.29; F = 44.30
7
7.5
8
8.5
9
9.5
10
-50 -40 -30 -20
Scorepol (Kcal/mol)
pEC
50
pEC50 = 5.96 (0.29) – 0.068 (0.0087) Scorepol – 0.014 (0.0033) Scoreapol n = 35; r2 = 0.72; q2 = 0.67; s = 0.24; F = 42.06
7.2
7.7
8.2
8.7
9.2
7.2 7.7 8.2 8.7 9.2pEC50 calculated
pEC
50 e
xper
imen
tal
Conclusions
The fragmental approach allows to obtain good models avoiding the construction of bovine rhodopsin clones.
The computational results are confirmed by the good correlation between the experimental data and the docking scores.
The method was successfully applied to the non-GPCR protein EAAT1, obtaining results confirmed by the experimental data.
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