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Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click-chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

The reaction conditions of the sortase-mediated ligation are examined, which can be used in the synthesis of a peptide nucleic acid-cell-penetrating peptide chimera. The newly obtained conjugates have displayed dose-dependent antisense activity.

The most affine corticotropin-releasing factor (CRF) receptor agonist were identified by photoaffinity cross-linking using p-benzoyl-L-phenylalanine (Bpa) analogues of urocortin (Ucn), interaction domains of CRF(sub 1) receptor with Bpa residues at exclusive positions. Results indicate that the very N-terminal ligand residues (1-11), which are responsible for receptor activation, are oriented to the juxtamembrane domain by interaction of amino acid residues 12, 17 and 22.

Isothermal titration calorimetry and fluorescence spectroscopy analyses reveal that a single amino acid substitution mediated charge change from a negative charge to a positive charge in the calcium-dependent binding site of calcodulin converts it into a target site for apocalmodulin, recognized by nitric oxide synthase I peptides. Furthermore, the substitution accompanied hydrophobicity enhances the apparent binding affinity.

The destabilization of residues 11 through 24 in helix I of amyloid beta protein causes the protein to form a beta sheet structure and aggregate into amyloid fibrils. This process occurs in the brains of patients with Alzheimer's disease.

Results show that the conformational behavior of the rat mitochondrial rhodanese presequence peptide, during its binding to small and large unilamellar phospholipid vesicles, is similar with respect to helix formation thermodynamics as determined by varying helix content.

Research has been conducted on the antibacterial peptide PGLa that exerts its activity by permeabilizing bacterial membranes. Experiments include the use of solid-state deuterium nuclear magnetic resonance.

Glutamic-acid-rich proteins (GARPs) exist as GARP1 and GARP2, and as a large cytoplasmic domain of the beta-subunit of the cyclic GMP-gated channel. GARPs have been identified as multivalent proteins interacting with those involved in cGMP signalling, phosphodiesterase and guanylate cyclase, and with a retina-specific ATP-binding cassette transporter.

Research was conducted to examine the water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. Conformational analyses suggest differing potentials of the various peptides to adopt the beta-sheet conformation. Both the influence of D-amino acid substitution and the length of the secondary structure domain were correlated with the beta-sheet stability and the association behavior. Results demonstrate that strong association of beta-strands leads to fibrillar structures which can bind Congo Red and exhibit birefringence.

A series of 1-hydroxy-7-azabenzotriazole (HOAt)-based coupling reagents was utilized for the cyclization reaction of all-L-pentapeptides to determine the efficiency of the reagents in increasing the reaction rate. The head-to-tail cyclization of all-L-penta- and -hexapeptides without turn-inducing amino acids was significantly improved by HOAt-derived onium and phosphonium salts. However, the reagents did not inhibit the formation of cyclodimers and C-terminal epimerization.

Investigation of the influence of the angle subtended by the positively charged helix face on membrane activity and selectivity of amphiphatic, antibacterial peptides found that the peptides's ability to induce dye release from negatively charged phosphatidyglycerol vesicles decreased with increasing angles. Results show that a large angle and a high content of negatively charged phospholipid favored binding. Data indicate that efficiency of analogs to permeate membranes is reduced by the electrostatic repulsion between adjacent helices in the pore.

The effects of peptide hydrophobicity on the binding abilities of magainin 2 (M2a) amide with phospholipid vesicles were analyzed via minimal sequence modifications. Circular dichroism analysis of the conformation of M2awith altered hydrophobicity in the vesicle-bound state indicated the presence of alterations in peptide binding affinity. Structural changes also affected peptide specificity for negatively charged lipid bilayers and modulated the activity of M2a against membranes with high levels of zwitterionic phospholipids.

A study used a method involving the measurement of the resulting intermolecular fluorescence energy transfer to investigate the association of lipid-bound magainin. One of the results showed that in the presence of a 10-fold excess of Nalpha-dansyl-magainin-2-amide, the Trp-Mag fluorescence intensity is reduced by roughly 20% compared to that of Trp-Mag in the absence of the acceptor. Experimental materials, methodology and findings are presented.

The use of several 1-hydroxy-7-azabenzotriazole-based onium salts in the cyclization process for thymopentin-derived pentapeptides showed azabenzotriazole-derived coupling agents to be most effective compared with traditional coupling reagents. Rapid cyclizations were observed, while monocyclic pentapeptide ring formation was favored by residual products of C-terminal D-amino acid.

An amphipathic model peptide, KLALKLALKALKAAKLA-NH2, and its complete double D-amino acid replacement set was utilized to examine the process of peptide binding at lipid vesicles of different surface charge and to determine the structure of the lipid-bound peptides using CD spectroscopy. Findings indicated that hydrophobic interactions drive the penetration of the amphipathic peptide structure into the inner membrane region, hence disturbing the arrangement of the lipid acyl chains and causing local disruption.

Systematic double-D-amino acid replacement of adjacent amino acids are utilized to investigate the secondary structure of the amphiphilic, antibiotic peptide magainin 2 amide (M2a) by circular dichroism spectroscopy. The secondary structure has a weak alpha-helix in the N-terminus and a stable alpha-helix between residues 9 and 21. The absence of conformational differences in the peptide when bound to vesicles of varying negative charge density, suggests a substantial independence of the structure from electrostatic forces.

The binding of calmodulin (CaM) to macrophage nitric oxide synthase type II (NOS-II) is regulated exclusively by the CaM-binding segment of the protein. Surface plasmon resonance spectroscopy studies indicate that the CaM binding affinities of neuronal nitric oxide synthase type I (NOS-I) and NOS-II are different. The binding of CaM to NOS-II is almost irreversible while NOS-I binds CaM reversibly with nanomolar affinities. CaM is bound to a small sequence of 25 amino acids in NOS-II.

1H NMR and circular dichroism spectroscopical studies using L-18 amphipathic alpha-helical peptide and its double D-amino acid analogue finds that the amalgamation of two D-amino acids causes variation only in the structure and flexibility. This makes the amino acid replacement a useful method for analyzing the helical conformation of biologically active peptides.

Fmoc amino acid fluorides act as coupling reagents in the stepwise solid phase synthesis of peptaibols, as proved by the syntheses of saturnisporin SA III, alamethicin F30 and F50 and trichotoxin A50J. The end products possess a high degree of purity in this reaction, in which several difficulties encountered in the earlier methods of synthesis, such as chain degradation and racemization, are overcome. The assembly of C-terminal peptide alcohols through O-chlorotrityl resin is the initial step of this successful method of synthesizing peptaibols.

Solid-phase approach is used to prepare medium-sized peptides with the help of Fmoc amino acid fluorides. These fluorides have the ability to couple two sterically hindered amino acid groups. Fmoc function is stable toward the base in the active acylating agent Fmoc-Aib-F. Efficiency of this method is proved by the synthesis of different peptides having hindered Aib units.