Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.

Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.

Non-specific binding and general cross-reactivity of Y receptor agonists are correlated and should importantly depend on their acidic sectors.

Non-specific binding of Y receptor agonists to intact CHO cells, and to CHO cell or rat brain particulates, is much greater for human neuropeptide Y (hNPY) compared to porcine peptide Y (pPYY), and especially relative to human pancreatic polypeptide (hPP). This binding of hNPY is reduced by alkali cations in preference to non-ionic chaotrope urea, while the much lower non-specific binding of pPYY is more sensitive to urea. The difference could mainly be due to the 10-16 stretch in 36-residue Y agonists (residues 8-14 in N-terminally clipped 34-peptides), located in the sector that contains all acidic residues of physiological Y agonists. Anionic pairs containing aspartate in the 10-16 zone could be principally responsible for non-specific attachments, but may also aid the receptor site binding. Two such pairs are found in hNPY, one in pPYY, and none in hPP. The hydroxyl amino acid residue at position 13 in mammalian PYY and PP molecules could lower conformational plasticity and the non-selective binding via intrachain hydrogen bonding. The acidity of this tract could also be important in agonist selectivity of the Y receptor subtypes. The differences point to an evolutionary reduction of promiscuous protein binding from NPY to PP, and should also be important for Y agonist selectivity within NPY receptor group, and correlate with partial agonism and out-of group cross-reactivity with other receptors.

Studies of the molecular mechanism of caspase-8 activation by solution NMR.

Caspases are the key players of apoptosis and inflammation. They are present in the cells as latent precursors, procaspases, and are activated upon an apoptotic or inflammatory stimulus. The activation mechanism of caspases has been studied extensively by biochemical and biophysical methods. Additional structural information on active caspases with a variety of different inhibitors bound at the active site is available. In this study, we investigated the cleavage mechanism of caspase-8 from its zymogen to active caspase-8 by solution NMR and by biochemical methods. The intermolecular cleavage reaction using the catalytically inactive C285A procaspase-8 mutant is triggered by adding caspase-8 and followed by (15)N,(1)H-NMR spectroscopy. The spectrum that initially resembles the one of procaspase-8 gradually over time changes to that of caspase-8, and disappearing peaks display exponential decaying intensities. Removal of either one of the cleavage recognition motifs in the linker, or phosphorylation at Tyr380, is shown to reduce the rate of the cleavage reaction. The data suggest that dimerization repositions the linker to become suitable for intermolecular processing by the associated protomer. Furthermore, analysis of inhibitor binding to the active caspase-8 reveals an induced-fit mechanism for substrate binding.

Biophysical and biological studies of end-group-modified derivatives of Pep-1.

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.

N-nitrosylation potential of mono-N-desethylamiodarone at physiological pH.

Amiodarone (AMI) is frequently used for the treatment of supraventricular arrhythmias. The parent drug is rapidly dealkylated to mono-N-desethylamiodarone (MDEA) and the plasma concentrations of AMI and MDEA are comparable. MDEA is a secondary amine and may thus undergo formation to the corresponding N-nitrosamine in combination with coadministered nitrovasodilators. Previous studies have shown that nitrovasodilators release the vasoactive NO? which may nitrosylate thiol or secondary amine groups in aqueous solutions. Therefore, the nitrosylation potential of MDEA at physiological pH was investigated. N-Nitroso-monodesethylamiodarone (NO-MDEA) was synthesized, characterized and used as a reference product for the detection of the corresponding N-nitrosamine. HPLC and NMR results have shown that the NO-MDEA product is an equilibrium of two configurational isomers (syn and anti). NO-release was generated by sodium nitroprusside (SNP) which was exposed to light. The formation to NO-MDEA was assayed by HPLC-UV. It has been found that MDEA is nitrosylated in the higher nanomolar range and that varying oxygenation of the reaction mixture did not significantly affect the reaction yields. The addition of thiols such as serum albumin (0.6mM), l-cysteine (2.5mM) or N-acetylcysteine (2.5mM) inhibited the NO-MDEA formation indicating that they may prevent N-nitrosamine formation in vivo. However, as S-nitrosothiols may also release NO?, in long term exposure to elevated levels of nitric oxide the nitrosylation of secondary amines may be taken into account.

A rationally designed oligopeptide shows significant conformational changes upon binding to sulphate ions.

Oligopeptides that interact with oxoanions were developed by rational design methods. The substrate-binding site of the enzyme purine nucleoside phosphorylase served as a model for the design of the ionophores. The amino acids involved in the complexation of oxoanions were linked through flexible spacer residues. These spacers were chosen such that the relative orientation of the interacting amino acids was conserved. Several peptide sequences were preselected based on intermolecular H-bond frequencies. These frequencies were calculated from molecular dynamics trajectories of the corresponding peptide-anion complexes and used to score the binding properties of the peptides. The most promising peptides were prepared using solid phase peptide synthesis. Anion binding of the peptide ionophores was screened using circular dichroism (CD) and confirmed by NMR spectroscopy. CD measurements performed in methanol revealed a significant conformational change of a linear undecapeptide upon binding to sulphate ions. Two-dimensional-NMR experiments confirmed that a conformation with high helical content is formed in the presence of sulphate ions. These conformational changes induced by the anion stimulate the development of new transduction mechanisms in chemical sensors.

Two novel cyclic peptides with antifungal activity from the cyanobacterium Tolypothrix byssoidea (EAWAG 195).

Two novel cyclic tridecapeptides, tolybyssidins A (1) and B (2), were isolated from the culture medium of mass cultured cyanobacterium Tolypothrix byssoidea (EAWAG 195) by means of bioguided isolation. The gross structures of these peptides were determined by 1D and 2D NMR experiments and tandem mass spectrometry. Both peptides contain the nonnatural amino acid dehydrohomoalanine (Dhha) as well as proteinogenic amino acids albeit with D- or L-configuration. The compounds exhibit moderate antifungal activity against the yeast Candida albicans.

New prenylated bi- and tricyclic phloroglucinol derivatives from Hypericum papuanum.

Five new prenylated tricyclic and three new bicyclic acylphloroglucinol derivatives have been isolated by bioactivity-guided fractionation of the petroleum ether extract of the dried aerial parts of Hypericum papuanum. The tricyclic compounds (1--5) were named papuaforins A--E. The bicyclic compounds were isolated together with their corresponding tautomers and were named hyperguinones A and B (6/6a,7/7a) and hyperpapuanone (8/8a), respectively. Their structures were elucidated on the basis of extensive 1D and 2D NMR experiments, as well as mass spectrometry. Furthermore, the cytotoxicity toward KB nasopharyngeal carcinoma cells and the antibacterial activity of the isolated compounds were determined.

NMR-restrained docking of a peptidic inhibitor to the N-terminal domain of the phosphoenolpyruvate:sugar phosphotransferase enzyme I.

Starting from the NMR structure of the binary complex between the N-terminal domain of the unphosphorylated enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase (PTS) and the histidine-containing phosphocarrier protein (HPr), a molecular model of the phosphorylated transition state of the related complex was established using constrained simulated annealing. The coordinates of the phosphorylated EIN enzyme were then used in a second step for flexible docking of a decapeptide inhibitor of EIN whose enzyme-bound conformation itself was determined by NMR using transferred nuclear Overhauser effects. Two phosphorylation models of the peptide inhibitor were investigated and shown to be both functional. Interestingly, one model is very similar to that of the complex between EIN and its natural substrate HPr. The present study demonstrates that NMR-guided flexible docking constitutes an interesting tool for docking highly flexible peptide ligands and facilitates the upcoming protein-based design of nonpeptide EIN inhibitors for discovering new antibiotics.

Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection.

The biological importance of the neuropeptide Y (NPY) has steered a number of investigations about its solution structure over the last 20 years. Here, we focus on the comparison of the structure and dynamics of NPY free in solution to when bound to a membrane mimetic, dodecylphosphocholine (DPC) micelles, as studied by 2D (1)H NMR spectroscopy. Both, free in solution and in the micelle-bound form, the N-terminal segment (Tyr1-Glu15) is shown to extend like a flexible tail in solution. This is not compatible with the PP-fold model for NPY that postulates backfolding of the flexible N terminus onto the C-terminal helix. The correlation time (tau(c)) of NPY in aqueous solution, 5.5 (+/-1.0) ns at 32 degrees C, is only consistent with its existence in a dimeric form. Exchange contributions especially enhancing transverse relaxation rates (R(2)) of residues located on one side of the C-terminal helix of the molecule are supposed to originate from dimerization of the NPY molecule. The dimerization interface was directly probed by looking at (15)N-labeled NPY/spin-labeled [TOAC34]-[(14)N]-NPY heterodimers and revealed both parallel and anti-parallel alignment of the helices. The NMR-derived three-dimensional structure of micelle-bound NPY at 37 degrees C and pH 6.0 is similar but not identical to that free in solution. The final set of 17 lowest-energy DYANA structures is particularly well defined in the region of residues 21-31, with a mean pairwise RMSD of 0.23 A for the backbone heavy atoms and 0.85 A for all heavy atoms. The combination of NMR relaxation data and CD measurements clearly demonstrates that the alpha-helical region Ala18-Thr32 is more stable, and the C-terminal tetrapeptide becomes structured only in the presence of the phosphocholine micelles. The position of NPY relative to the DPC micelle surface was probed by adding micelle integrating spin labels. Together with information from (1)H,(2)H exchange rates, we conclude that the interaction of NPY with the micelle is promoted by the amphiphilic alpha-helical segment of residues Tyr21-Thr32. NPY is located at the lipid-water interface with its C-terminal helix parallel to the membrane surface and penetrates the hydrophobic interior only via insertions of a few long aliphatic or aromatic side-chains. From these data we can demonstrate that the dimer interface of neuropeptide Y is similar to the interface of the monomer binding to DPC-micelles. We speculate that binding of the NPY monomer to the membrane is an essential key step preceeding receptor binding, thereby pre-orientating the C-terminal tetrapeptide and possibly inducing the bio-active conformation.

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein.

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.

Degradation mechanism and stability of 5-aminolevulinic acid.

The physiological substance and precursor of the heme synthesis 5-aminolevulinic acid (ALA) is a promising prodrug for photodiagnosis and photodynamic therapy of epithelial tumors, particularly in urological and gynecological tissues. For the clinical use of this substance, a chemically stable and sterile drug formulation is required. In the present study, degradation mechanism of ALA in aqueous solution and possibilities to improve its stability were examined. A capillary electrophoretic method was developed that was suitable for the quantification of ALA and of two degradation products. The intermediate degradation product was 2, 5-dicarboxyethyl-3,6-dihydropyrazine, which was further oxidized to 2,5-dicarboxyethylpyrazine. The structures of the degradation products were proven by (1)H and (13)C nuclear magnetic resonance spectroscopy. ALA degradation was very efficiently inhibited by adjusting the pH of the aqueous solution to a value <5 and by purging with nitrogen. Additives such as antioxidants did not improve the ALA stability. These results demonstrated that low pH ALA aqueous solution may be one possible dosage form to be considered for market introduction.

The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats.

The first Y(5) receptor-selective analog of neuropeptide Y (NPY), [Ala(31),Aib(32)]NPY, has been developed and biologically characterized. Using competition binding assays on cell lines that express different Y receptors, we determined the affinity of this analog to be 6 nm at the human Y(5) receptor, >500 nm at the Y(1) and Y(2) receptors, and >1000 nm at the Y(4) receptor. Activity studies performed in vitro using a cAMP enzyme immunoassay, and in vivo using food intake studies in rats, showed that the peptide acted as an agonist. Further peptides obtained by the combination of the Ala(31)-Aib(32) motif with chimeric peptides containing segments of NPY and pancreatic polypeptide displayed the same selectivity and even higher affinity (up to 0.2 nm) for the Y(5) receptor. In vivo administration of the new Y(5) receptor-selective agonists significantly stimulated feeding in rats. The NMR solution structures of NPY and [Ala(31),Aib(32)]NPY showed a different conformation in the C-terminal region, where the alpha-helix of NPY was substituted by a more flexible, 3(10)-helical turn structure.

In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres.

The purpose of this study was to design poly(lactide-co-glycolide) (PLGA) microspheres for the continuous delivery of the somatostatin analogue, vapreotide, over 2-4 weeks. The microspheres were produced by spray-drying and the desired characteristics, i.e. high encapsulation efficiency and controlled release over 2-4 weeks, achieved through optimizing the type of polymer, processing solvent, and co-encapsulated additive. The in vitro release was tested in fetal bovine serum preserved with 0.02% of thiomersal. Furthermore, formulations were injected intramuscularly into rats to obtain pharmacokinetic profiles. Encapsulation efficiency was between 34 and 91%, depending on the particular formulation. The initial peptide release (within 6 h) was lowest, i.e. <20%, when acetic acid was used as processing solvent and highest, i.e. 57%, with dichloromethane. The various co-encapsulated additives generally lowered the encapsulation efficiency by 15-30%. The best formulation in terms of low burst and effective drug serum levels (>1 ng/ml) over 21-28 days in rats was the one made with end-group uncapped PLGA 50:50, the solvent acetic acid and the additive polyethyleneglycol. In conclusion, the optimization of formulation parameters allowed us to produce vapreotide-loaded PLGA microspheres of suitable characteristics for therapeutic use.

New phloroglucinol derivatives from Hypericum papuanum.

Bioactivity-guided fractionation of the petroleum ether extract of the aerial parts of Hypericum papuanum led to the isolation of five new tricyclic phloroglucinol derivatives. On the basis of extensive 1D and 2D NMR experiments as well as MS studies, their structures were elucidated as the C-3 epimers of 8-hydroxy-4,4, 7-trimethyl-9-(2-methylpropionyl)-3-(1-methylvinyl)-5beta -H-tricyclo[ 5.3.1.0(1,5)]undec-8-ene-10,11-dione (1,2); the C-3 epimers of 8-hydroxy-4,4, 7-trimethyl-9-(2-methylbutyryl)-3-(1-methylvinyl)-5beta-H-++ +tricyclo[5. 3.1.0(1,5)]undec-8-ene-10,11-dione (3, 4), and 8-hydroxy-4,4, 7-trimethyl-9-(2-methylpropionyl)-5beta-H-tricyclo[5.3 .1.0(1, 5)]undec-8-ene-10,11-dione (5), and their corresponding tautomers (1a, 2a, 3a, 4a, 5a). Compounds 1/1a-5/5a were named ialibinones A-E, respectively. Compounds 1/1a-4/4a showed antibacterial activity against Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus.

Evidence for a dynamic structure of human neuronal growth inhibitory factor and for major rearrangements of its metal-thiolate clusters.

Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.

Iridoid glycosides of Leonurus persicus.

Two new iridoid glucosides, 6-O-acetylajugol (1) and 7, 8-epoxy-8-epi-loganic acid (2), together with five known iridoid glucosides, galiridoside (3), ajugoside (4), 10-deoxygeniposidic acid (5), 7-deoxy-8-epi-loganic acid (6), and 8-O-acetylharpagide (7), have been isolated from the aerial parts of Leonurus persicus. Leucosceptoside A (8), eugenyl beta-rutinoside (9), and kaempferol 3-O-glucoside (10) were also isolated. The structures of 1 and 2 were elucidated by extensive 1D- and 2D-NMR spectroscopy and molecular modeling. The structure of 3 was confirmed by single-crystal X-ray diffraction. Antimicrobial activity of compounds (1-10) was also evaluated against a panel of gram-positive and gram-negative bacteria and two strains of fungi.

The NMR solution conformation of unligated human cyclophilin A.

The nuclear magnetic resonance (NMR) solution structure of free, unligated cyclophilin A (CypA), which is an 18 kDa protein from human T-lymphocytes that was expressed in Escherichia coli for the present study, was determined using multidimensional heteronuclear NMR techniques. Sequence-specific resonance assignments for 99.5% of all backbone amide protons and non-labile hydrogen atoms provided the basis for collection of an input of 4101 nuclear Overhauser enhancement (NOE) upper distance constraints and 371 dihedral angle constraints for distance geometry calculations and energy minimization with the programs DIANA and OPAL. The average RMSD values of the 20 best energy-refined NMR conformers relative to the mean coordinates are 0.49 A for the backbone atoms and 0.88 A for all heavy atoms of residues 2 to 165. The molecular architecture includes an eight-stranded antiparallel beta-barrel that is closed by two amphipathic alpha-helices. Detailed comparisons with the crystal structure of free CypA revealed subtle but significant conformational differences that can in most cases be related to lattice contacts in the crystal structure. 15N spin relaxation times and NMR lineshape analyses for CypA in the free form and complexed with cyclosporin A (CsA) revealed transitions of polypeptide loops surrounding the ligand-binding site from locally flexible conformations in the free protein, some of which include well-defined conformational equilibria, to well-defined spatial arrangements in the CypA-CsA complex. Compared to the crystal structure of free CypA, where the ligand-binding area is extensively involved in lattice contacts, the NMR structure presents a highly relevant reference for studies of changes in structure and internal mobility of the binding pocket upon ligand binding, and possible consequences of this conformational variability for calcineurin recognition by the CypA-CsA complex.

Modified, cyclic dodecapeptide analog of neuropeptide Y is the smallest full agonist at the human Y2 receptor.

In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100-fold increase in affinity compared to the C-terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS-KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH-SY5Y cells and cyclic [Lys28-Glu32] NPY Ac-25-36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D-NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.