Molecular Interaction between Distal C-Terminal Domain of the CB1 Cannabinoid Receptor and Cannabinoid Receptor Interacting Proteins (CRIP1a/CRIP1b).

We have investigated the structure of the distal C-terminal domain of the of the CB1 cannabinoid receptor (CB1R) to study its interactions with CRIP1a and CRIP1b using computational techniques. The amino acid sequence from the distal C-terminal domain of CB1R (G417-L472) was found to be unique, as it does not share sequence similarity with other protein structures, so the structure was predicted using ab initio modeling. The computed model of the distal C-terminal region of CB1R has a helical region between positions 441 and 455. The CRIP1a and CRIP1b were modeled using Rho-GDI 2 as a template. The three-dimensional model of the distal C-terminal domain of the CB1R was docked with both CRIP1a as well as CRIP1b to study the crucial interactions between CB1R and CRIP1a/b. The last nine residues of CB1R (S464TDTSAEAL4722) are known to be a CRIP1a/b binding site. The majority of the key interactions were identified in this region, but notable interactions were also observed beyond theses nine residues. The multiple interactions between Thr418 (CB1R) and Asn61 (CRIP1a) as well as Asp430 (CB1R) and Lys76 (CRIP1a) indicate their importance in the CB1R-CRIP1a interaction. In the case of CRIP1b, multiple hydrogen bond interactions between Asn437 (CB1R) and Glu77 (CRIP1b) were observed. These interactions can be critical for CB1R's interaction with CRIP1a/b, and targeting them for further experimental studies can advance information about CRIP1a/b functionality.

CB1 cannabinoid receptor-phosphorylated fourth intracellular loop structure-function relationships.

A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.

In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) - CB1 cannabinoid receptor.

Cannabinoid Receptor Interacting Protein isoform 1b (CRIP1b) is known to interact with the CB1 receptor. Alternative splicing of the CNRIP1 gene produces CRIP1a and CRIP1b with a difference in the third exon only. Exons 1 and 2 encode for a functional domain in both proteins. CRIP1a is involved in regulating CB1 receptor internalization, but the function of CRIP1b is not very well characterized. Since there are significant identities in functional domains of these proteins, CRIP1b is a potential target for drug discovery. We report here predicted structure of CRIP1b followed by its interaction analysis with CB1 receptor by in-silico methods A number of complementary computational techniques, including, homology modeling, ab-initio and protein threading, were applied to generate three-dimensional molecular models for CRIP1b. The computed model of CRIP1b was refined, followed by docking with C terminus of CB1 receptor to generate a model for the CRIP1b- CB1 receptor interaction. The structure of CRIP1b obtained by homology modelling using RHO_GDI-2 as template is a sandwich fold structure having beta sheets connected by loops, similar to predicted CRIP1a structure. The best scoring refined model of CRIP1b in complex with the CB1 receptor C terminus peptide showed favourable polar interactions. The overall binding pocket of CRIP1b was found to be overlapping to that of CRIP1a. The Arg82 and Cys126 of CRIP1b are involved in the majority of hydrogen bond interactions with the CB1 receptor and are possible key residues required for interactions between the CB1 receptor and CRIP1b.

Membrane-induced structure of novel human tachykinin hemokinin-1 (hHK1).

PPT-C encoded hemokinin-1(hHK-1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK-1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three-dimensional structure of the hemokinin-1 in aqueous and micellar environment has been studied by one and two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin-1 was unstructured in aqueous environment; anionic detergent SDS induces α-helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3-M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK-1-NK1 interaction that is essential for hHK1 based signaling events.

Molecular recognition of tachykinin receptor selective agonists: insights from structural studies.

This Review deals essentially with the elucidation of structural features of Tachykinin family of neuropeptides, which are known to interact through three distinct GPCR subtypes, namely NK1 (Neurokinin 1), NK2 (Neurokinin 2) and NK3 (Neurokinin 3) receptors. In mammals, Tachykinins have been shown to elicit a wide array of activities such as powerful vasodilatation, hypertensive action and stimulation of extravascular smooth muscle and are known to be involved in a variety of clinical conditions including chronic pain, Parkinson's disease, Alzheimer's disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma. This broad spectrum of action of Tachykinins is attributed to the lack of selectivity of tachykinins to their receptors. All tachykinins interact with all the three-receptor subtypes with SP preferring NK1, NKA preferring NK2 and NKB preferring NK3. This lack of specificity can be accounted for by the conformational flexibility of these short, linear peptides. Hence, identification of structural features of the agonists important for receptor binding and biological activity is of great significance in unraveling the molecular mechanisms involved in tachykinin receptor activation and also in rational design of novel therapeutic agents. Understanding structure of the ligand-receptor complex and analysis of topography of the binding pocket of the tachykinin receptor is also crucial in rational design of drugs.

Molecular modeling of neurokinin B and tachykinin NK3 receptor complex.

The tachykinin receptor NK3 is a member of the rhodopsin family of G-protein coupled receptors. The NK3 receptor has been regarded as an important drug target due to diverse physiological functions and its possible role in the pathophysiology of psychiatric disorders, including schizophrenia. The NK3 receptor is primarily activated by the tachykinin peptide hormone neurokinin B (NKB) which is the most potent natural agonist for the NK3 receptor. NKB has been reported to play a vital role in the normal human reproduction pathway and in potentially life threatening diseases such as pre-eclampsia and as a neuroprotective agent in the case of neurodegenerative diseases. Agonist binding to the receptor is a critical event in initiating signaling, and therefore a characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK3 has been developed. A three-dimensional model for the NK3 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from photoaffinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK3 receptor complex obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor.

Specific interaction of jacalin with phycocyanin, a fluorescent phycobiliprotein.

Recent research has shown that, like porphyrins, phycocyanin (PC) too can produce singlet oxygen upon excitation with the appropriate radiation and hence could be useful in photodynamic therapy (PDT) for cancer. Unlike porphyrins, PC has the advantage of being a non-toxic, non-carcinogenic, soluble protein. However, the challenge would be to target the fluorescent phycobiliprotein to malignant cells. We report here that the tumor-specific lectin, jacalin, binds PC specifically in a carbohydrate-independent manner and with affinities better than that for porphyrins. Hence the lectin could prove to be a useful carrier for targeted delivery of PC. The interaction involves both ionic and hydrophobic interactions and more than one contact site.

Molecular modeling of the peptide agonist-binding site in a neurokinin-2 receptor.

The neurokinin-2 receptor is a member of the rhodopsin family of G-protein coupled receptors, which represents one of the most relevant target families in small-molecule drug design. NK-2 receptors have been implicated in playing a pathophysiological role in asthma. Activation of the NK-2 receptor by its endogenous peptide agonist, tachykinins, is associated with diverse biological responses like bronchoconstriction, vasodepression, and regulation of endocrine functions. Agonist binding to the receptor is a crucial event in initiating signaling, and therefore characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKA with its G-protein coupled receptor neurokinin-2 receptor has been developed. A three-dimensional model for the NK-2 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKA to the receptor has been performed utilizing the ligand binding data obtained from the photoaffinity labeling and site-directed mutagenesis studies. The molecular model for the NKA/NK-2 receptor complex thus obtained sheds light on the topographical features of the binding pocket of the receptor and provides atomic insight into the biochemical data currently available for the receptor. The results of the receptor modeling studies have been used to discuss the molecular determinants for NK-2 receptor selectivity.

Three-dimensional structure of Phyllomedusin, a NK1 receptor agonist bound to dodecylphosphocholine micelles.

Phyllomedusin, an amphibian tachykinin decapeptide, has been shown to be selective for Neurokinin 1 receptor. Because the micelle-associated structure may be relevant to the Phyllomedusin-receptor interaction, the three-dimensional structure of the Phyllomedusin in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Sequence specific resonance assignments of protons have been made from correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using DYANA. The CD and NMR results show that, while in water Phyllomedusin prefers to be in an extended chain conformation, whereas in the presence of dodecylphosphocholine micelles, a membrane model system, a partial helical conformation is induced. Analysis of NMR data indicates that the global fold of Phyllomedusin can be explained in terms of equilibrium between 3(10)-helix and alpha-helix from residue 4 to 10. An extended highly flexible N-terminus displays some degree of order and a possible turn structure. A comparison between the conformational features of Phyllomedusin and different Neurokinin 1 receptor agonist indicates several common features in the distribution of hydrophobic and hydrophilic residues. The conformational similarities suggest that the molecules interact with receptor in an analogous manner.

Structural characterization of neurokinin-3 receptor selective peptide agonist scyliorhinin II bound to DPC micelles.

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12-18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.

Unique helical conformation of the fourth cytoplasmic loop of the CB1 cannabinoid receptor in a negatively charged environment.

The proximal portion of the C-terminus of the CB(1) cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401-417), the intracellular loop 4 (IL4) peptide, mimicked the receptor's G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3(10)-helical conformation is induced. The predominance of 3(10)-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB(1) receptor and might influence the signal to the G-proteins.

Solution structure of amphibian tachykinin Uperolein bound to DPC micelles.

Uperolein, a physalaemin-like endecapeptide, has been shown to be selective for Neurokinin 1 receptor. As a first step towards understanding the structure-activity relationship, we report the membrane-induced structure of Uperolein with the aid of circular dichroism and 2D (1)H NMR spectroscopy. Sequence-specific resonance assignments of protons have been made using correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using torsion angle molecular dynamics within program DYANA. The conformational range of the peptide revealed by NMR and CD studies has been analysed in terms of characteristic secondary features. Analysis of NMR data indicates that the global fold of Uperolein can be explained in terms of equilibrium between 3(10)-helix and alpha-helix from residues 5 to 11. An extended highly flexible N-terminus displays some degree of order and a possible turn structure. A comparison between the structures of Uperolein and Substance P, a prototype and endogenous Neurokinin 1 receptor agonist, indicates several common features in the distribution of hydrophobic and hydrophilic residues. Both the peptides show an amphiphilic character towards the middle region. The similarities suggest that the molecules interact with the receptor in an analogous manner.

Neuroprotective role of neurokinin B (NKB) on beta-amyloid (25-35) induced toxicity in aging rat brain synaptosomes: involvement in oxidative stress and excitotoxicity.

The brain tissue has a large oxidative capacity, but its ability to combat oxidative stress is limited. In aging brain tissue the oxidative stress increases due to decreased activity of antioxidant enzymes and increased oxidative stress leading to neurodegeneration associated with excitotoxicity. The aim of the present study was to determine the effect of neuropeptides, neurokinin B (NKB) and amyloid beta protein fragment Abeta (25-35) and neurotransmitters N-methyl D-aspartate (NMDA) and Glutamate on rat brain synaptosomes of different age groups. Aging brain functions were assessed by measuring the activities of superoxide dismutase (Mn-SOD) and monoamine oxidase (MAO) and intrasynaptosomal [Ca(2+)](i )levels in presence of neuropeptides and neurotransmitters. Increase in age decreased the SOD and MAO enzyme activities; Abeta (25-35) addition further had damaging/toxic effects on the enzymes, whereas NKB alone and in combination with amyloid lowered the toxic effects caused by Abeta (25-35) addition, which was concentration (peptide) and age dependent. Oxidative stress and excitotoxicity are major consequences associated with the age, [Ca(2+)](i )was increased with the age and the neuropeptides and neurotransmitters elicited significant modulatory effects on it. Our study elucidates an increased activity of SOD, decreased activity of MAO and restoration of [Ca(2+)](i) levels in the presence of NKB and suggests an antioxidant, neuromodulatory and neuroprotective role of tachykinin peptide NKB against the beta amyloid induced toxicity.

Membrane associated functions of neurokinin B (NKB) on Abeta (25-35) induced toxicity in aging rat brain synaptosomes.

The effect of different concentrations (0.1-5 microM) of neurokinin B (NKB) and Abeta (25-35) on acetylcholine esterase (AChE), Na(+)-K(+) ATPase and membrane fluidity (DPH anisotropy) were investigated in rat brain synaptosomes of 3, 9, 18 and 30 months old. An age dependent decrease was observed for all the three parameters studied. An in vitro incubation of isolated brain synaptosomes with Abeta (25-35) showed toxic effects on all the parameters studied and the peptide had concentration and age dependent effects, while NKB showed stimulating effect on the parameters and the combined NKB+Abeta (25-35) incubations showed a partial reversal effect as compared to the Abeta (25-35) alone. Thus, the results suggest a membrane mediated function for NKB and its role in neuromodulation, neuroprotection and antioxidant property against Abeta (25-35) induced toxicity in aging brain functions.

Three-dimensional structure of neuropeptide k bound to dodecylphosphocholine micelles.

Neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), represents the most potent and longest lasting vasodepressor and cardiomodulatory tachykinin reported thus far. NPK has been shown to have high selectivity for the NK2 receptor. Because the micelle-associated structure may be relevant to the NPK-receptor interaction, the three-dimensional structure of the NPK in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The interproton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment NPK lacks a definite secondary structure, although some turn-like elements are present in the N terminus. The structure is well-defined in the presence of dodecylphosphocholine micelles. The global fold of NPK bound to DPC micelles consists of two well-defined helices from residues 9 to 18 and residues 27 to 33 connected by a noncanonical beta turn. The N terminus of the peptide is characterized by a 3(10) helix or a series of dynamic beta turns. The conformational range of the peptide revealed by NMR and circular dichroism (CD) studies has been analyzed in terms of characteristic secondary features. The observed conformational features have been further compared to a NKA and neuropeptide gamma (NPgamma) potent endogenous agonist for the NK2 receptor.

Membrane-Induced Structure of Scyliorhinin I: A Dual NK1/NK2 Agonist.

Scyliorhinin I, a linear decapeptide, is the only known tachykinin that shows high affinity for both NK-1 and NK-2 binding sites and low affinity for NK-3 binding sites. As a first step to understand the structure-activity relationship, we report the membrane-induced structure of scyliorhinin I with the aid of circular dichroism and 2D-(1)H NMR spectroscopy. Sequence specific resonance assignments of protons have been made from correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using DYANA. The superimposition of 20 final structures has been reported with backbone pairwise root mean-square deviation of 0.38 +/- 0.19 A. The results show that scyliorhinin I exists in a random coil state in aqueous environments, whereas helical conformation is induced toward the C-terminal region of the peptide (D4-M10) in the presence of dodecyl phosphocholine micelles. Analysis of NMR data is suggestive of the presence of a 3(10)-helix that is in equilibrium with an alpha-helix in this region from residue 4 to 10. An extended highly flexible N-terminus of scyliorhinin I displays some degree of order and a possible turn structure. Observed conformational features have been compared with respect to that of substance P and neurokinin A, which are endogenous agonists of NK-1 and NK-2 receptors, respectively.

Effect of hormone replacement therapy in normalizing age related neuronal markers in different age groups of naturally menopausal rats.

Aging of the normal brain is accompanied by changes in its structure, function, and metabolism. There are significant gender differences in aging brain. Most of these changes increase during menopausal condition in females when the level of estradiol and progesterone are decreased. The objective of this study was to determine the effect of estradiol and progesterone (separate as well as combined) hormones in neuronal tissues from naturally menopausal rats of different age groups. Results show decreased activity of Acetylcholine esterase (AChE) whereas the level of lipid peroxidation increased with age, and after the hormone treatments both AChE activity and level of lipid peroxidation returned to control values. The deposition of lipofuscin, a pigment that accumulated intraneuronally in brain and other tissues and is considered a marker of aging, was increased with aging and the hormone treatment decreased this deposition. The present study clearly shows reduction in risk factors associated with aging in the murine model system by hormone treatments, namely estrogen and progesterone by increasing the activity of acetylcholine esterase and decreasing the levels of lipid peroxidation and lipofuscin deposition in different parts of aging brain. This study suggests that hormone replacement therapy may either reduce or delay the onset of age related diseases like Alzheimer's, Parkinson's and other neurological disorders.

Estradiol and progesterone treatments change the lipid profile in naturally menopausal rats from different age groups.

The effect of estradiol and progesterone therapy in serum and liver on the lipid profile of naturally menopausal albino rats of the Wistar strain of different age groups (12,18 and 24 months) have been measured and compared with the age matched groups. Three months old rats were used as young controls. The aged rats were administered subcutaneous injection of 17-beta-estradiol (0.1 microg/g body weight), progesterone (2.5 microg/g body weight) and similar concentrations of both in combined treatment for 1 month and the level of triglycerides (TG), total lipids (TL), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were measured in serum and liver of 3, 12, 18 and 24 months old control as well as treated groups. The results show that TG, HDL, VLDL levels were increased significantly by 71%, 155%, 54%, respectively in liver of 24 months old rats by combination treatment when compared with age matched control animals. The levels of TL, TC and LDL were decreased by 20%, 31%, and 30%, respectively in serum of 12 months old rats in combination treatment group. The effect was more significant in 12 and 24 months old female rats with administration of estrogen and combined (EP) treatments. The results indirectly suggest that hormone replacement therapy (HRT) can reduce the risk of cardiovascular disease (CVD) thereby playing a cardio-protective role by restoring lipid and hormone levels to the similar levels as found in young female animals.

Membrane-induced structure of the mammalian tachykinin neuropeptide gamma.

Neuropeptide gamma (NPgamma) is a neurokinin-2 (NK-2) receptor selective agonist, which plays an important role in mediation of asthma and elicits a wide range of biological responses like bronchoconstriction, vasodepression and regulation of endocrine functions. The structure determination of this peptide agonist is important in understanding the molecular basis of peptide ligand recognition by the receptor and for rational drug design. In the present study we report the solution structure of NPgamma characterized by circular dichroism (CD) spectropolarimetry and 2D (1)H NMR spectroscopy in both aqueous and membrane mimetic solvents. Effect of calcium ions on the conformation of NPgamma was also studied using CD spectropolarimetry. Sequence-specific resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NPgamma prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region of the peptide (K13-M21) in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system. A type II' beta turn from H9 to R11 precedes the helical core in the C-terminus of NPgamma. N-terminus of NPgamma also displays some degree of order and a possible turn structure. Conformation adopted by NPgamma in presence of lipid micelles represents a structural motif typical of NK-2 selective agonists and is similar to that observed for Neurokinin A in hydrophobic environment. The observed conformational features have been correlated to the binding ability and biological activity of NPgamma.

Three dimensional structure of mammalian tachykinin peptide neurokinin B bound to lipid micelles.

Neurokinin B (NKB), a decapeptide of mammalian origin exhibits a variety of biological activities such as regulatory functions in reproduction, pre-eclampsia and neuroprotection in Alzheimer's disease. In order to gain insight into structure-function relationship, three-dimensional structure of NKB has been investigated using CD spectropolarimetry and two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy in aqueous and membrane mimetic solvents. Unambiguous NMR assignments of resonances have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and Nuclear Overhauser Effect Spectroscopy (NOESY) experiments. Distance constraints obtained from the NMR data have been used to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Our data show that a helical structure is induced in NKB, in presence of perdeuterated dodecyl phosphocholine (DPC) micelles, a membrane model system. Further, the conformation adopted by NKB in presence of DPC micelles represents a structural motif typical of neurokinin-3 selective agonists.