ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 349, No. 2, January 15, pp. 225–235, 1998 Article No. BB970395 Conformational Changes of Neuromedin B and Delta Sleep-Inducing Peptide Induced by Their Interaction with Lipid Membranes as Revealed by Spectroscopic Techniques and Molecular Dynamics Simulation 1 Eugenia Polverini,* Rita Casadio,* Paolo Neyroz,† and Lanfranco Masotti† ,2 *Laboratory of Biophysics, Department of Biology, and †Section of Pharmaceutical Biochemistry, Department of Biochemistry ‘‘G. Moruzzi,’’ University of Bologna, 40127 Bologna, Italy Received July 7, 1997, and in revised form September 15, 1997 favor the correct peptide – receptor contact and recog- nition. For DSIP, the lipid-stabilized conformation Static and dynamic spectroscopic properties of the does not support an amphiphilic structure-driven pep- tryptophanil emission in conjunction with circular di- tide – membrane interaction and suggests a hydropho- chroism (CD) spectroscopy and molecular dynamics bicity-driven diffusion across the bilayer.  1998 Academic are used to investigate the interactions of the neuro- Press peptide neuromedin B (NMB) and the membrane-per- Key Words: neuropeptides; time-resolved fluores- meable d sleep-inducing peptide (DSIP) with the mem- cence; molecular dynamics; peptide conformation; brane lipid phase. Our data indicate that in solution lipid membranes. both peptides exist in energetically equivalent confor- mations, whereas in the presence of the membrane specific conformational states are stabilized. By Recent studies have demonstrated that small pep- changing from the aqueous to the lipid phase, the tides are able to exist in dynamic equilibria between static and the dynamic fluorescence properties of the unfolded and folded structures, depending on the sol- NMB’s tryptophan residue are clearly affected: the vent polarity and/or their interaction with the mem- fluorescence steady-state spectrum as well as the re- brane phase (1–3). This is so for a variety of neuro- solved fluorescence decay-associated spectra (DAS) transmitters, peptides, and hormones for which the im- are shifted to the blue with a significant increase of portance of the ordered structures has been recognized the fluorescence intensity of the second lifetime com- in relation to binding to G protein-coupled membrane ponent (t 2 -DAS). On the other hand, in the lipid envi- receptors (4). Furthermore, when small peptides per- ronment the same parameters of DSIP are negligibly meate the membrane to fulfil physiological require- affected as compared to the aqueous buffer. The CD ments, a certain folding may be required for a favorable and molecular dynamics analyses are consistent with interaction with the lipid moiety (5). these results and indicate that, while NMB assumes a A major question to be answered is therefore to what helix-like conformation with the tryptophan residue extent the membrane phase is affecting peptide confor- in the apolar surface, DSIP adopts a globule-like struc- mation and whether function can be related to a given ture with the indole ring that is surface-exposed. As structure. In order to investigate this issue, in the pres- previously found for neuromedin C (Polverini, E., Neyroz, P., Fariselli, P., Casadio, R., and Masotti, L., ent paper we analyze the effects of the membrane Biochem. Biophys. Res. Commun. 214, 663–668, 1995), phase in promoting a stable conformation of two small for NMB the stabilized ‘‘lipophilic’’ structure also may peptides: neuromedin B (NMB) 3 and the d sleep-induc- 3 Abbreviations used: CD, circular dichroism; DAS, decay-associ- 1 This work was supported by a M.U.R.S.T. 60% grant to L.M. and ated spectra; DMPC, L-a-dimyristoyl-phosphatidylcholine; DSIP, d sleep-inducing peptide; EDTA, ethylenediaminetetraacetic acid; a CNR grant to R.C. 2 To whom correspondence should be addressed at Section of Phar- LPC, L-a-lysophosphatidylcholine; NMB, neuromedin B; NMC, neu- romedin C; PBS, phosphate-buffered saline; PS, L-a-phosphatidyl-L- maceutical Biochemistry, Department of Biochemistry ‘‘G. Moruzzi,’’ Via S. Donato, 19/2, 40127 Bologna, Italy. Fax: 39-51-242978. E- serine; rmsd, root mean square deviation; TFE, trifluoroethanol; Tris, tris(hydroxymethyl)aminomethane. mail: masotti@biocfarm.unibo.it. 225 0003-9861/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.