Multiple 6-Bromotryptophan Residues in a Sleep-Inducing Peptide † Elsie C. Jimenez, ‡,# Maren Watkins, § and Baldomero M. Olivera* ,‡ Departments of Biology and Pathology, UniVersity of Utah, Salt Lake City, Utah 84112, and Department of Physical Sciences, College of Science, UniVersity of the Philippines Baguio, Baguio City, Philippines ReceiVed May 24, 2004; ReVised Manuscript ReceiVed July 13, 2004 ABSTRACT: We have characterized a novel sleep-inducing peptide comprising 33 amino acids with three residues of the unusual posttranslationally modified amino acid, 6-bromotryptophan. The peptide, termed “light sleeper” or the r7a conotoxin, was purified from the venom of the fish-hunting Conus radiatus. The light sleeper peptide has additional notable biochemical properties; it equilibrates slowly between two distinct conformers, and has four γ-carboxyglutamate residues. The pattern of posttranslational bromination in the light sleeper peptide suggests that tryptophan residues at N- and C-termini may be preferential sites for posttranslational bromination. We describe the isolation and characterization of a peptidic gene product with the highest number of posttranslationally modified tryptophan residues yet characterized. The peptide has other unusual biological and biophysical features: it is sleep-inducing, and it equilibrates slowly between two different conformations. The bromination of tryptophan (to 6-bromotryptophan) was only recently established to be a posttranslational modification (1, 2) (for a review, see ref 3). Natural products that seem to be derived from 6Br-Trp are not uncommon in the marine environment. However, it was the characterization of Conus peptides with 6Br-Trp and the accompanying demonstration that these were bona fide gene products directly translated from mRNA that firmly established that bromination is a true posttranslational modification. Initially, this might have been regarded as an esoteric adaptation in a highly specialized biological system. More recently however, an effort to characterize ligands for orphan G-protein-coupled receptors led two different laboratories to demonstrate that this posttranslational modification occurs in a neuropeptide from mammalian brain (4, 5). Unusual posttranslational modifications have often been initially characterized in highly specialized biological sys- tems, and then subsequently shown to be much more widely distributed. One classic example is the γ-carboxylation of glutamate to γ-carboxyglutamate (Gla), 1 which for many years was thought to be a specialization of the vertebrate blood clotting cascade. Some years later, this posttranslational modification was shown to occur in Conus peptides. Fur- thermore, it appears from more recent biochemical and molecular work that the γ-carboxylation enzyme machinery is expressed in a variety of mammalian tissues (suggesting diverse physiological roles), and that the relevant modifica- tion enzyme, γ-glutamyl carboxylase, is found in Drosophila and Anopheles, as well as in vertebrate systems and Conus (6, 7). Thus, the demonstration that bromination occurs in both the Conus peptide system and in mammalian brain raises the strong possibility that this posttranslational bromination is similarly likely to be much more widely distributed in biology. For such posttranslational modifications, it would be of use to predict when they may occur. With genomic technology being ever more widely used, the possibility becomes ever greater that posttranslationally modified gene products will be missed when using conventional molecular analyses. The ability to pinpoint when posttranslational modification may occur has been key to the rapid develop- ment of such fields as protein phosphorylation and N- glycosylation. To begin to recognize potential sites for posttranslational modification, it is first necessary to identify actually modified amino acids from native modified gene products in as many different sequence contexts as possible. The peptide characterized in this report is highly unusual in that it contains three different Br-Trp residues, more than in any other gene product characterized so far. Thus, it could provide one important guidepost for predicting when tryp- tophan residues may be brominated. Indeed, the pattern of bromination we have found suggests that closer scrutiny for potential posttranslational bromination in certain mammalian neuropeptides is justified. Additionally, the peptide described here is noteworthy in that it induces a sleep-like state in mice. Pharmacological agents that induce sleep are potential tools for understanding central nervous system function. This peptide was isolated from the venom of a cone snail, Conus radiatus, that has three unrelated peptides that all induce sleep (2, 8). Each † This work was supported by a Program Project from the National Institutes of Health, GM 48677. * Corresponding author: Baldomero M. Olivera, Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, Utah, 84112; telephone (801) 581-8370; fax (801) 585-5010; e-mail olivera@biology.utah.edu. ‡ Department of Biology, University of Utah. § Department of Pathology, University of Utah. # University of the Philippines Baguio. 1 Abbreviations: 6Br-Trp, 6-bromotryptophan; Gla, γ-carboxy- glutamate; ACN, acetonitrile; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography; MALDI, matrix assisted laser desorption ionization; DTT, dithiothreitol; PTH, phenylthiohydantoin; Tris, tris-hydroxymethylaminomethane; PCR, polymerase chain reac- tion; UTR, untranslated region; EST, expressed sequence tag; NMDA, N-methyl-D-aspartate. 12343 Biochemistry 2004, 43, 12343-12348 10.1021/bi0489412 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/03/2004