The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: Synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies
The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: Synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies(349 views) De Luca S, Ragone R, Bracco C, Digilio G, Tesauro D, Saviano M, Pedone C, Morelli G
Keywords: Cck8 Binding, Ccka Receptor, Fluorescence, Cholecystokinin Receptor, Desulfocholecystokinin Octapeptide, Amino Terminal Sequence, Article, Binding Affinity, Calculation, Circular Dichroism, Complex Formation, Controlled Study, Fluorescence Spectroscopy, Membrane Stabilization, Micelle, Molecular Dynamics, Nuclear Overhauser Effect, Priority Journal, Protein Conformation, Protein Interaction, Protein Secondary Structure, Protein Synthesis, Proton Nuclear Magnetic Resonance, Receptor Binding, Structure Analysis, Titrimetry, Humans, Magnetic Resonance Spectroscopy, Models, Peptide Fragments, Protein Binding, Sincalide, Solvents,
Affiliations: *** IBB - CNR ***
Ctr. Interuniv. Ric. Peptidi Bioat., Ist. di Biostructure e Bioimmagini, CNR, via Mezzocannone 6-8, 80134 Napoli, Italy
Dipto. di Biochimica e Biofisica, Seconda Università di Napoli, 80138 Napoli, Italy
Bioindustry Park del Canavese, 10010 Colleretto Giacosa, Italy
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The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: Synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies
The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinin receptor, CCKA-R(32-47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCKA-R(32-47) encompassing residues 39-46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N-terminal region (segment 32-37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCKA-R(32-47) was different from that found for the intact N-terminal receptor tail, CCKA-R(1-47). To assess whether CCKA-R(32-47) was still able to bind the nonsulfated cholecystokinin C-terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCKAR(1-47) and CCK8. The different affinities for the ligand shown by CCKA-R(32-47) and CCKA-R(1-47) were paralleled by different interaction modes between the receptor segments and the micelles. The interaction of CCKA-R(32-47) with DPC micelles. was much weaker than that of CCKA-R(1-47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCKA-R(32-47) was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1-31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N-terminal extracellular tail of the CCKA receptor. Copyright (C) 2003 European Peptide Society and John Wiley Sons, Ltd.
The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: Synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies
The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: Synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies