Aggregation Behaviour and Zn2+ Binding Properties of Secretin

Abstract

Dynamic light scattering measurements were carried out on secretin in aqueous solution (2 mM; pH 5.0). The results indicated that the molecule exists as a fairly compact hexamer under these solution conditions. Secondary structural properties of the secretin hexameric complex were evaluated using CD and NMR spectroscopy. Specifically, the spectral properties of secretin in water were examined as a function of peptide concentration. Results from the analyses indicated a 2-fold increase (17−32%) in α-helical content within the region Ser¹¹−Arg²¹ as the peptide concentration was increased from 0.1 to 2 mM. Displacement of the αH proton chemical shifts relative to random coil values did not alter significantly with increasing peptide concentration. This observation confirmed that the length of the helical segment is independent of peptide concentration between 0.1 and 2 mM. The nature of the helix was furthermore determined as amphipathic, and thus the potential for a cooperative intermolecular association through the apolar helical face of individual monomers was indicated. These findings suggest that secretin aggregates into symmetric hexamers at millimolar concentrations and, furthermore, that the helical domain is stabilized through this intermolecular association. The potential for secretin to bind divalent cations, including Ca²⁺ and Zn²⁺, was also examined by CD¹ and NMR spectroscopy. The results revealed that Zn²⁺ specifically coordinates to the His¹ and Asp³ residues of each secretin monomer without disrupting the peptide's helical structure, whereas Ca²⁺ did not exhibit any interaction with the peptide hormone. It was concluded from these studies that secretin may be stored in a hexameric form within its secretory tissues and that zinc may play a role in the storage of secretin through a specific interaction with the N-terminal histidine and aspartic acid residues.