Polymers of N-substituted glycines (''peptoids'') containing chiral centers at the ␣ position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of Ϸ6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.Polymers of N-substituted glycines, termed peptoids, form a new class of biocompatible, synthetically accessible heteropolymers. Their sequence-specific, automated, and highly efficient synthesis has allowed the creation of combinatorial libraries of peptoid oligomers for drug discovery (1-3). By using recent improvements in the efficiency of the coupling chemistry, peptoids up to 50 residues in length have been synthesized. Among those, cationic peptoid 36-mers have been identified that bind DNA, protect the DNA from nuclease digestion, and facilitate gene transfection (4).We recently showed that many peptoids with chiral centers at the side chain ␣ position have strong CD signals, indicating the presence of a regularly repeating conformation, in both aqueous and organic solvents (5). This structure is remarkably stable, as demonstrated by both CD and differential scanning calorimetry (DSC) measurements (5). A model of this conformation was recently proposed based on the results of molecular mechanics and semi-empirical quantum mechanical calculations (6). Modeling predicted that peptoids containing (S)-N-(1-phenylethyl)glycine residues would form righthanded helices with a periodicity of approximately three residues per turn and cis-amide bonds, similar to the polyproline type I conformation. In this conformation, the backbone carbonyls are aligned along the long helical axis, providing an explanation for the intensity of the helical CD signal.Presented here is the structure determination by NMR spectroscopy of the major solution isomer of compound 1, a pentapeptoid containing five chiral side chains (Fig. 1A). ...

Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc Parathyroid hormone (PTH)1 is the major regulator of calcium levels in blood and plays a role in the regulation of bone remodeling (1). Given intermittently, PTH displays anabolic activity in bone and, therefore, has considerable therapeutic potential (2). PTH and PTH-related protein exert their actions via a seven-transmembrane (TM) domain-containing receptor (PTH1-Rc) (3) belonging to a subfamily of related G proteincoupled receptors (4 -11). The PTH1-Rc is coupled to both adenylyl cyclase/cyclic AMP and phospholipase C/inositol 1,4,5-trisphosphate/cytosolic calcium intracellular signaling pathways (12)(13)(14)(15).Understanding the molecular mechanism of ligand recognition and signal transduction by the PTH1-Rc may identify new directions for the design of novel hormone analogs for the treatment of diseases such as osteoporosis, hypercalcemia of malignancy and hyperparathyroidism (16). In order to directly identify the structural elements involved in PTH-PTH1-Rc interactions, we employed a photoaffinity scanning approach (17). The generation of covalently linked ligand-receptor conjugates and the identification of the cross-linked domains allows mapping of the interface between hormone and receptor. Photoaffinity cross-linking has been successfully applied in defining interactions between small peptides, such as substance P (18 -20), cholecystokinin (21), and vasopressin (22), and their receptors. Recently, we used this general approach to identify directly the interaction between position 13 of PTH and a 17-amino acid domain (residues 173-189) of the hPTH1-Rc (17).We now report the evaluation of a series of photoreactive analogs obtained by a "p-benzoylphenylalanine (Bpa) scan" of the principal receptor activation domain (residues 1-6) of 34)), maintained full potency and led to the identification of a second "contact domain" between PTH and hPTH1-Rc. This information allows us to create, for the first time, a model describing interactions of hPTH-(1-34) with its receptor based on direct identification of the interacting regions. EXPERIMENTAL PROCEDURESMaterials-Boc-protected amino acids, N-hydroxybenzotriazole, N,NЈ-dicyclohexylcarbodiimide, and p-methylbenzydrylamine resin were purchased from Applied Biosystems (Foster City, CA). Boc-(3-iodo)tyrosine ] was from Peninsula Laboratories (Belmont, CA). B&J brand dichloromethane, N-methylpyrrolidone, and acetonitrile were obtained from Baxter (McGraw Park, IL). IODOGEN ® and 2-(2Ј-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole) were purchased from Pierce. Cyanogen bromide was from Aldrich. Na

The bimolecular complex of the C-terminal octapeptide of cholecystokinin, CCK-8, with the N-terminus of the CCK(A)-receptor, CCK(A)-R(1-47), has been structurally characterized by high-resolution NMR and computational refinement. The conformation of CCK(A)-R(1-47), within the lipid environment used for the spectroscopic studies, consists of a well-defined alpha-helix (residues 3-9) followed by a beta-sheet stabilized by a disulfide linkage between C18 and C29, leading to the first transmembrane alpha-helix (TM1). Titration of CCK(A)-R(1-47) with CCK-8 specifically affects the NMR signals of W39 of the receptor, in a saturable fashion. This association is specific for CCK-8; no association was observed upon titration of CCK(A)-R(1-47) with other peptide hormones. The ligand/receptor complex was characterized by intermolecular NOEs between Tyr(27) and Met(28) of CCK-8 and W39 of CCK(A)-R(1-47). These findings suggest that CCK-8 binds to CCK(A) with the C-terminus within the seven-helical bundle and the N-terminus of the ligand, projecting out between TM1 and TM7, forming specific interactions with the N-terminus of the CCK(A) receptor. This mode of ligand binding, consistent with published mutagenesis studies, requires variation of the interpretation of recent findings from photoaffinity cross-linking studies.

Parathyroid hormone (PTH) regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renal tubular cells and is fully functional as the N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped" conformation with Nand C-terminal helices brought in close proximity by a turn has been postulated. The general acceptance of this hypothesis, despite limited experimental evidence, has altered the direction of the design of PTH-analogs. Examining the structure of human PTH-(1-34) under conditions that encompass the different environments the hormone may experience in the approach to and interaction with the G-protein-coupled receptor (including benign aqueous and saline solutions and in the presence of dodecylphosphocholine), we observe no evidence for a U-shape conformation or any tertiary structure. Instead, the N-and C-terminal helical domains, which vary in length and stability depending on the condi-

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The N-methylation of cyclic peptides can be used to modify the activity and/or selectivity of biologically active peptides. As N-methylation introduces different flexibility and lipophilicity, it can also improve the bioavailability (the ADMET profile). To search for conformationally constrained cyclic peptides, a library of 30 different N-methylated peptides with the basic sequence cyclo(-D-Ala-L-Ala4-) was synthesized. Based on the NMR analysis, seven of these peptides exhibited single conformations (>98%). The structural features of these peptides were determined by a combination of NMR and distance geometry and then further refined by molecular dynamics simulations in an explicit DMSO solvent box. The structures provided from these efforts can now serve as templates for the rational design of cyclic pentapeptides with a distinct backbone conformation or for "spatial screening" to explore the bioactive conformation of medically important peptide systems.

Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Gai and Gaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg 10 -kallidin or desArg 9 -BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-a. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and selfmaintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews

The fully active cholecystokinin analog (Thr,Nle)-CCK-9 was lipo-derivatized by N-terminal grafting of a dimyristoylglycerol moiety to induce tight interdigitation with cell membrane bilayers. While the parent CCK peptide was shown to interact only transiently with small unilamellar phospholipid vesicles, the lipo-CCK peptide, although self-aggregating into vesicles, inserts rapidly and quantitatively into phospholipid bilayers. Fluorescence and, even more so, NMR data are supportive for a chain reversal of the CCK moiety of the lipo derivative with embedment of the C-terminus into hydrophobic compartments of the bilayer. MD simulations allowed for a proposal of the folded form of CCK in bilayers with a helical array parallel to the interface and an amphipathic display of the side chains. In this model, the phenylalanine aromatic ring is heading the peptide molecule and may thus play a decisive role in the lateral penetration of the receptor at the water/lipid interface. In fact, despite the membrane-bound state, its binding affinity for rat pancreatic acini is comparable to that of the CCK peptide when tested after a 3-h equilibration period but 5-6-fold lower at 45 min, suggesting that the association rate is significantly lower than that of the unmodified CCK peptide. This can rationally be attributed to the tight interdigitation of the double-tailed lipo moiety with the membrane bilayer. Moreover, an escape of the lipopeptide into the extracellular aqueous phase is energetically highly unfavored; therefore, the receptor can only be reached by a membrane-bound two-dimensional migration. The observed difference in amplification between binding and amylase secretion may result from inadequate occupation of low-affinity CCK receptors, which leads then to poor couplings to G-proteins. Nevertheless the data confirm that lateral penetration of receptor structures is possible, and thus, preadsorption of peptide (neuro)hormones at the cell membrane bilayer may indeed represent the first step in the receptor recognition process.