Conformational analysis of cholecystokinin CCK26-33 and related fragments by 1H NMR spectroscopy, fluorescence-transfer measurements, and calculations.
The conformational behavior of CCK7, Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, and CCK8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, in their sulfated and unsulfated forms, was studied both by 1H NMR spectroscopy in dimethyl-d6 sulfoxide and water and by fluorescence-transfer measurements at pH 7. In neutral conditions, both experimental methods show that these peptides exist preferentially in folded forms with beta and gamma turns around the sequence Gly-Trp-Met-Asp and Met-Asp-Phe-NH2, respectively. The presence of stable folded conformations is supported by through-space effects during the titration of the ionizable groups and by the weak temperature dependency of some amide protons not only in dimethyl sulfoxide but also in water. The folding of the C-terminal part, already shown in CCK5, seems to be a common conformational characteristic in CCK peptides. The N-terminal part of CCK8 presents an equilibrium between beta and gamma turns, whereas this part of the peptide is more flexible in CCK7. The low quantum yield of Tyr and the large mean distance (R = 15 A) between Tyr and Trp, determined by fluorescence-transfer measurements, support the occurrence of folded conformations pushing the aromatic rings far from each other. Interestingly, the introduction of the sulfate group enhances the folding tendency even in aqueous medium. The larger amide temperature dependency and the decrease in the R distance at acidic pH suggest that an intramolecular ionic interaction involving the N-terminal amino group and the beta-carboxyl groups of Asp32 stabilize the folded forms. Metropolis calculations performed on CCK8 support the existence of stable folded conformations closely related to those deduced from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D002766:Cholecystokinin / Q000031:analogs & derivatives*; D004735:Energy Transfer; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010446:Peptide Fragments; D011487:Protein Conformation; D012844:Sincalide / Q000031:analogs & derivatives*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; C044201:cholecystokinin (26-33); D002766:Cholecystokinin; D012844:Sincalide