DELTA-SLEEP-INDUCING PEPTIDE - SOLUTION CONFORMATIONAL STUDIES OF A MEMBRANE-PERMEABLE PEPTIDE

Peptides and peptide-like molecules as a class have very poor permeability through biological membranes, which severely compromises their potential effectiveness as therapeutic agents. In order to gain insight into the problem of delivering peptide and protein drugs and to establish a model in which the effects of systematic structural variations on transport can be explored, an investigation of the solution conformation of a membrane-permeable peptide was undertaken. Delta-sleep-inducing peptide (DSIP, MW 849) was used in this investigation. DSIP is a charged, hydrophilic peptide that possesses the unusual ability to diffuse passively across the blood-brain barrier (BBB) in vivo [Kastin, A. J., Banks, W. A., Castellanos, P. F., Nissen, C., & Coy, D. H. (1982) Pharmacol. Biochem. Behav. 17, 1187-1191] and across monolayers of brain microvessel endothelial cells in vitro, a model of the BBB [Raeissi, S., & Audus, K. L. (1989) J. Pharm. Pharmacol. 41, 848-852]. This nonapeptide was studied in solution using one- and two-dimensional nuclear magnetic resonance (NMR), circular dichroism (CD), Fourier transform infrared (FT-IR), and fluorescence spectroscopies in conjunction with molecular modeling. Our spectroscopic findings suggest that DSIP exists in a dynamic equilibrium between unordered and folded structures. Residues 2-5 and 6-9 tend to form type I beta-turns in aqueous solution and a similar, but more ordered, helix-like structure inducible in 40% trifluoroethanol (TFE). NMR, FT-IR, and CD studies in aqueous solution support the dynamic equilibrium hypothesis with the IR data, suggesting that the beta-turn population is approximately 40%. A Trp(1) fluorescence maximum of 360 nm at pH 6.5 indicates that the indole ring is exposed to the solvent and not protected by the proposed folded structure. The fluorescence lifetime of Trp(1) also suggests that the indole ring in DSIP experiences an environment similar to that of free Trp in aqueous solution. The most ordered structure consistent with these data was one in which both proposed turns were of the beta I type. This model has a compact structure with several intramolecular hydrogen bonds and appears to be amphiphilic. The structure in 40% TFE suggested by NMR and CD studies was also examined by modeling. The helix-like model that fit the TFE data was very closely related to the structure in aqueous solution (energy difference = 2.5 kcal/mol). These studies suggest that compact structures that maximize intramolecular hydrogen bonding in aqueous solution may influence permeability due to the nature of the folded structure itself or that the folded structure is an energetically favorable precursor to the preferentially permeable conformation.