Abstract: The GAGA factor of Drosophila melanogaster uses a single Cys2-His2-type zinc finger for specific DNA binding. Comparative sequence alignment of the GAGA zinc finger core with other structurally characterized zinc fingers reveals that the beta-hairpin of the GAGA zinc finger prefers amino acids with an aliphatic side-chain different from those of other zinc fingers. To probe the substitution effect of aromatic amino acids in the beta-hairpin on the DNA binding, three mutant peptides were designed by substituting consensus phenylalanine, an aromatic amino acid, at key positions in the beta-hairpin region. The metal-binding and the overall fold of the mutant peptides are very similar to those of the wild-type as shown by UV-vis absorption spectroscopy and circular dichroism spectroscopy. However, the gel mobility shift assay and isothermal calorimetric studies demonstrated that none of the mutants are able to bind the cognate DNA substrate, although the mutation is confined only to the beta-hairpin region. The present results suggest that the nature of the amino acids in the beta-hairpin plays an important role in the DNA-binding of the GAGA factor protein.

Abstract: The design of artificial functional DNA-binding proteins has long been a goal for several research laboratories. The zinc finger proteins, which typically contain many fingers linked in tandem fashion, are some of the most studied DNA-binding proteins. The zinc finger protein's tandem arrangement and its the ability to recognize a wide variety of DNA sequences make it an attractive framework to design novel DNA-binding peptides/proteins. Our laboratory has utilized several design strategies to create novel zinc finger peptides by re-engineering the C(2)H(2)-type zinc finger motif of transcription factor Sp1. Some of the engineered zinc fingers have shown nuclease and catalytic functional properties. Based on these results, we present the design strategies for the creation of novel zinc fingers.

Abstract: To experimentally demonstrate the mirror-image recognition in protein and DNA interaction, we have designed a small DNA-binding peptide based on the zinc-finger domain of GAGA transcription factor. Circular dichroism data suggest that the conformations of peptide enantiomers as well as the DNA enantiomers have a mirror-image relationship. The gel mobility shift assay showed that the synthetic enantiomers of the peptide showed reciprocal chiral-specific interactions with the DNA; the natural L-peptide binds specifically with the natural D-DNA substrate, and the unnatural D-peptide binds specifically with the unnatural L-DNA substrate. The present data imply that the folding of the L- and D-enantiomers of the peptide as well as the DNA substrates are exact mirror images of each other in 3-D structure and biological activity, and generalize the chiral-specific nature of biomolecular interaction.

Abstract: A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K(D)) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).

Abstract: The application of endogenous neuropeptides (e.g., enkephalins) as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood-brain barrier (BBB). Effective BBB transport of glycosylated enkephalins has been demonstrated in several labs now. Analgesia (antinociception) levels greater than morphine, and with reduced side effects have been observed for several glycopeptides related to enkephalin. Somewhat paradoxically, enhanced BBB transport across this lipophilic barrier is achieved by attaching water-soluble carbohydrate groups to the peptide moieties to produce biousian glycopeptides that can be either water-soluble or membrane bound. Transport is believed to rely on an endocytotic mechanism (transcytosis), and allows for systemic delivery and transport of the water-soluble glycopeptides. Much larger endorphin/dynorphin glycopeptide analogs bearing amphipathic helix address regions also have been shown to penetrate the BBB in mice. This holds forth the possibility of transporting much larger neuropeptides across the BBB, which may encompass a wide variety of receptors beyond the opioid receptors.

Abstract: Antinociceptive effects and BBB transport properties of glycosylated enkephalin derivatives are reviewed. Previously, the application of enkephalins as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood brain barrier. This shortcoming has been overcome by glycosylation, paradoxically leading to enhanced BBB transport via transcytosis. Principal design considerations required for enhanced binding, stability and transport of opioid peptides are reviewed. Modifications of the peptide backbone and side chains to achieve optimal receptor binding (micro/delta-selectivity) are presented. The importance of reversible binding between the glycopeptide and membranes is emphasized, and several pertinent examples of peptide-membrane interactions are discussed in the light of glycopeptide transport and opioid binding. An "amphipathic hypothesis" is introduced as a rationale for the observed BBB penetration of the opioid glycopeptides.

Abstract: Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth and subsequent amino acid residues (Ser or Thr) and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono- or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared with their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55 degrees C tail-flick test. The calculated A(50) values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A (50) values ranging from 2.2 to 46.4 micromol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis- and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.

Abstract: A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.

Abstract: The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural analysis of CD81 has enabled the synthesis of small molecules designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compounds has been synthesized. Seven related, imidazole-based compounds were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compounds have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 associations with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81.

Abstract: Fumaramide derivatives were analyzed in solution by (1)H NMR spectroscopy and in the solid state by X-ray crystallography in order to characterize the formation of CH...O interactions under each condition and to thereby serve as models for these interactions in peptide and protein structure. Solutions of fumaramides at 10 mM in CDCl(3) were titrated with DMSO-d(6), resulting in chemical shifts that moved downfield for the CH groups thought to participate in CH...O=S(CD(3))(2) hydrogen bonds concurrent with NH...O=S(CD(3))(2) hydrogen bonding. In this model, nonparticipating CH groups under the same conditions showed no significant change in chemical shifts between 0.0 and 1.0 M DMSO-d(6) and then moved upfield at higher DMSO-d(6) concentrations. At concentrations above 1.0 M DMSO-d(6), the directed CH...O=S(CD(3))(2) hydrogen bonds provide protection from random DMSO-d(6) contact and prevent the chemical shifts for participating CH groups from moving upfield beyond the original value observed in CDCl(3). X-ray crystal structures identified CH...O=C hydrogen bonds alongside intermolecular NH...O=C hydrogen bonding, a result that supports the solution (1)H NMR spectroscopy results. The solution and solid-state data therefore both provide evidence for the presence of CH...O hydrogen bonds formed concurrent with NH...O hydrogen bonding in these structures. The CH...O=C hydrogen bonds in the X-ray crystal structures are similar to those described for antiparallel beta-sheet structure observed in protein X-ray crystal structures.