An approach to peptide-based ATP receptors by a combination of random selection, rational design, and molecular imprinting.
Random selection, rational design and molecular imprinting were cooperatively utilized to develop peptide-based ATP synthetic receptors. In this fusion strategy, combinatorial chemistry was utilized for screening a precursor peptide useful for construction of ATP receptors, and rational design was employed in modification of the selected precursor peptide for higher affinity and selectivity. Finally, molecular imprinting was used for pre-organizing the conformation of the precursor peptide as complementary to a target molecule ATP. The fusion strategy appeared to have advantage to sole use of the individual strategy: (1) a low hit-rate of combinatorial chemistry will be improved by customizing a higher order structure of a selected peptide by molecular imprinting, (2) combinatorial chemistry allows us to semi-automatically select components of water-compatible synthetic receptors, (3) rational design improves the selected peptide sequence for better molecularly imprinted receptors. A peptide consisting of a randomly selected sequence and a rationally designed sequence (Resin-Lys-Gly-Arg-Gly-Lys-Gly-Gly-Gly-Glu-Lys-Tyr-Leu-Lys-NHAc) was designed and synthesized as a precursor peptide. The rational design was made according to the sequence of the adenine binding site of biotin carboxylase. The on-beads peptide was cross-linked with dimethyl adipimidate in the presence of ATP. In the saturation binding tests, the cross-linked on-beads peptide showed 5.3 times higher affinity compared to the non-cross-linked peptide with the same sequence. Furthermore, the cross-linked peptide showed improved selectivity; the ratios of binding constants, K((ATP))/K((ADP)) and K((ATP))/K((GTP)), were increased from 2.4 to 19, and from 0.8 to 10, respectively. It would be notable that the peptide without the rationally designed sequence showed no discrimination between ATP and GTP (K((ATP))/K((GTP)) as 0.9), suggesting that the rationally designed site was successfully engaged for recognition of the adenine base.
Matsui
Jun
J
Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan. matsui@konan-u.ac.jp
Nagano
Junji
J
Miyoshi
Daisuke
D
Tamaki
Katsuyuki
K
Sugimoto
Naoki
N
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D015195:Drug Design; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D054802:Molecular Imprinting / Q000379:methods*; D015394:Molecular Structure; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000378:metabolism
D009842:Oligopeptides; D018048:Receptors, Purinergic P2; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate