Amino acid sequence of sweet-taste-suppressing peptide (gurmarin) from the leaves of Gymnema sylvestre.

The complete amino acid sequence of a sweet-taste-suppressing peptide, gurmarin, from the leaves of Gymnema sylvestre was determined by the Edman analysis of peptides derived from digests obtained with Staphylococcus aureus V8 protease, pyroglutamyl aminopeptidase, and lysyl endopeptidase. Gurmarin consists of 35 amino acid residues with an amino-terminal pyroglutamyl residue and has the molecular weight of 4,209. Gurmarin has no significant homology with other known proteins.     

Reduction of the suppressive effects of gurmarin on sweet taste responses by addition of beta-cyclodextrin

Cyclodextrins (CDs) have the remarkable ability to form inclusion complexes with a wide variety of guest molecules. In the present study, possible influences of CDs on gurmarin inhibition of the chorda tympani responses to sucrose were examined in C57BL mice. Responses to sucrose were suppressed to approximately 50% of control by treatment of the tongue with 30 micrograms/ml (approximately 7.1 microM) gurmarin. Rinsing the tongue with 15 mM beta-CD after gurmarin gave rapid recovery of the suppressed sucrose responses to approximately 85% of control, whereas 15 mM alpha- or gamma-CD did not. When gurmarin was mixed with beta-CD, the suppressive effects of gurmarin on sucrose responses were largely reduced. No such reduction was observed for mixtures with alpha- and gamma-CD. Gurmarin includes tyrosine and tryptophan residues whose aromatic rings are directed outward and can probably form inclusion complexes with beta-CD. Therefore, the observed reduction of the effects of gurmarin may be due to steric hindrances in inclusion complexes of gurmarin with beta-CD that may interfere with gurmarin binding to sweet taste receptors.      

Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris-HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.     

Inhibitory effect of gurmarin on palatal taste responses to amino acids in the rat

Gurmarin (10 microg/ml), a protein extracted from Gymnema sylvestre, depressed significantly (40-50%) the phasic taste responses to sugars (sucrose, fructose, lactose, and maltose) and saccharin sodium recorded from the greater superficial petrosal nerve (GSP) innervating palatal taste buds in the rat. However, no significant effect of gurmarin was observed for taste responses to NaCl, HCl, and quinine hydrochloride. Phasic responses to D-amino acids that taste sweet to humans (His, Asn, Phe, Gln) were also depressed, but gurmarin treatment was without significant effect on taste responses to D-Trp and D-Ala, six L-amino acids (His, Asn, Phe, Gln, Trp, and Ala), and two basic amino acid HCl salts (Arg and Lys). With the exception of D-Trp, these inhibitory effects of gurmarin on GSP taste responses were related to the rat's preference for these substances.     

Investigation of the solution conformation of cytochrome c-551 from Pseudomonas stutzeri.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri (ATCC 17588). Resonance assignments are proposed for all main-chain and most side-chain protons. Distance constraints were determined on the basis of nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scaler coupling constants. Twenty-four structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1033 interproton distance and 57 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for a loop region around residues 34-40, the first two residues at the N-terminus and the last two at the C-terminus, and some side chains located on the molecular surface. The average root mean squared deviation in position for equivalent atoms between the 24 individual structures and the mean structure obtained by averaging their coordinates is 0.54 +/- 0.08 A for the main-chain atoms and 0.97 +/- 0.09 A for all non-hydrogen atoms of residues 3-80 plus the heme group. These structures were compared to the X-ray crystallographic structure of an analogous protein, cytochrome c-551 from Pseudomonas aeruginosa [Matsuura, Takano, & Dickerson (1982) J. Mol. Biol. 156, 389-409). The main-chain folding patterns are very consistent, but there are some differences. The largest difference is in a surface loop segment from residues 34 to 40.     

Location of the Disulfide Bonds of the Sweetness-suppressing Polypeptide Gurmarin

The sweetness-suppressing polypeptide gurmarin has been isolated from the leaves of Gymnema sylvestre and consists of 35 amino acid residues including three intramolecular disulfide bonds. The primary structure has already been determined. The positions of the disulfide bonds were located, by a combination of mass spectrometric analysis and sequencing of cystine-containing pep tides obtained by thermolysin-catalyzed hydrolysis of gurmarin, to be at Cys3–Cys18, Cys10–Cys23, and Cys17–Cys33.     

Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.     

Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers

Activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoK(ATP) channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoK(ATP) channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 +/- 0.02 at baseline to 0.50 +/- 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoK(ATP) channels, which increased the IC(50) for ATP from 335 +/- 4 to 940 +/- 34 microM at 0.8 mM (P < 0.05, n = 5 approximately 8). We conclude that direct activation of the mitoK(ATP) channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning.     

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

Beta-cyclodextrin inhibits the sweet taste suppressing activity of gurmarin by the formation of an inclusion complex with aromatic residues in gurmarin.

Our recent study in mice revealed that the inhibitory activity of gurmarin on the sweet taste responses was reduced significantly by the presence of beta-cyclodextrin (beta-CD). To investigate the mechanism involved in the action of beta-CD, physicochemical experiments were performed on the interaction of CDs with gurmarin examining the effect of CDs on the UV absorption spectrum of gurmarin and on the elution behavior in gel filtration (or exclusion) chromatography. Among the three kinds of cyclodextrins tested, beta-CD induced significant changes in the UV absorption spectrum of gurmarin that were characteristic of those found in the inclusion complex formation of tyrosine and tryptophan with beta-CD. The abnormal retention behavior of gurmarin in gel filtration resulting from hydrophobic interaction with the gel matrix reverted to normal in the presence of beta-CD in the elution buffer. These results suggest that the unique domain of gurmarin, in which five aromatic amino acid residues are all directed outwardly and form a hydrophobic cluster, is a possible site of interaction with the gurmarin-sensitive sweet taste receptor molecules in rodents.     

Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

Induction of salivary kallikreins by the diet containing a sweet-suppressive peptide, gurmarin, in the rat

Gymnema sylvestre (gymnema) contains gurmarin that selectively inhibits responses to sweet substances in rodents. The present study investigated possible interaction between gurmarin and the submandibular saliva in rats fed diet containing gymnema. Electrophoretic analyses demonstrated that relative amounts of two proteins in the saliva clearly increased in rats fed the gymnema diet. However, rats previously given section of the bilateral glossopharyngeal nerve showed no such salivary protein induction. Analyses of amino acid sequence indicate that two proteins are rat kallikrein 2 (rK2) and rat kallikrein 9 (rK9). rK2 and rK9, a family of serine proteases, have a striking resemblance of cleavage site in the protein substrates. Interestingly, gurmarin possesses comparable residues with those rK2 and rK9 prefer. The kallikreins significantly inhibited immunoreaction between gurmarin and antigurmarin antiserum. These results suggest that rK2 and rK9 increased by chemosensory information for the gymnema diet via the glossopharyngeal nerve might cleave gurmarin or at least cause specific binding with it.     

The conformation of deamino-oxytocin: X-ray analysis of the 'dry' and 'wet' forms

Two crystal structures of (1 beta-mercaptopropionic acid) deamino-oxytocin are reported. The 'dry form' in space group C2 has cell dimensions a = 27.08 +/- 0.03, b = 9.06 +/- 0.01, c = 22.98 +/- 0.02 A, beta = 102.06 +/- 0.03 with one deamino-oxytocin and six water molecules per asymmetric unit. The 'wet form' in space group P2(1) has cell dimensions a = 27.27 +/- 0.02, b = 9.04 +/- 0.01, c = 23.04 +/- 0.02 A, beta = 102.24 +/- 0.02, with two deamino-oxytocin and 13 water molecules per asymmetric unit. A local twofold parallel to the monoclinic axis gives a pseudo C2 packing. Initial phases of the 'dry form' were calculated by the heavy-atom method from the isomorphous and anomalous difference Pattersons and anomalous difference Fouier synthesis. The structure was refined by using restrained least-squares at 1.2 A resolution to a crystallographic R = 0.10. The molecular replacement method yielded the P2(1) structure that was refined with geometric restraints to R less than 0.09, by using all data to 1.09 A resolution. Deamino-oxytocin consists of a cyclic tocin ring formed by six amino acids, closed by a disulphide bridge, S1-S6, and held by two trans-annular hydrogen bonds N2-O5 and N5-O2 with a type II turn at residues 3 and 4. A flexible tripeptide tail has a loosely hydrogen-bonded type I beta-turn between N9 and O6. The sulphur of cysteine at position 1 is disordered in all the molecules leading to alternative hands of disulphide. The conformational flexibility of Ile 3, Asn 5, Pro 7 side chains and the disulphide bridge is consistent with previous models of oxytocin in which flexibility is necessary for biological activity.     

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 1H NMR spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 11 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.3 A for residues 2-39 and 0.9 +/- 0.2 A for residues 5-37. The corresponding values for all atoms are 1.9 +/- 0.3 and 1.4 +/- 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N- (residues 1-4) and C- (residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.     

The polypeptide fold of the globular domain of histone H5 in solution. A study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The polypeptide fold of the 79-residue globular domain of chicken histone H5 (GH5) in solution has been determined by the combined use of distance geometry and restrained molecular dynamics calculations. The structure determination is based on 307 approximate interproton distance restraints derived from n.m.r. measurements. The structure is composed of a core made up of residues 3-18, 23-34, 37-60 and 71-79, and two loops comprising residues 19-22 and 61-70. The structure of the core is well defined with an average backbone atomic r.m.s. difference of 2.3 +/- 0.3 A between the final eight converged restrained dynamics structures and the mean structure obtained by averaging their coordinates best fitted to the core residues. The two loops are also well defined locally but their orientation with respect to the core could not be determined as no long range ([i-j[ greater than 5) proton-proton contacts could be observed between the loop and core residues in the two-dimensional nuclear Overhauser enhancement spectra. The structure of the core is dominated by three helices and has a similar fold to the C-terminal DNA binding domain of the cAMP receptor protein.     

Characterization of SNP, a novel tissue- and phase-specific nuclear protein expressed during the proliferative phase in the oviduct of the lizard Podarcis sicula Raf

The amino acid sequence of a novel tissue-and phase-specific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 +/- 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the C-terminus. They have thus been named [des-(Ala81) SNP1] and [des-(Lys80-Ala81) SNP2], respectively. The molecular weights are 9140.21 +/- 0.83 for [des-(Ala81) SNP1] and 9011 +/- 0.09 for [des-(Lys80-Ala81) SNP2].     

Synthesis,characterization, and sweetness-suppressing activities of gurmarin analogues missing one disulfide bond

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and includes three intramolecular disulfide bonds. The roles of the three disulfide bonds were investigated by replacing each with two alanine residues by solid-phase synthesis. Nine analogues of [Ala^3,18]gurmarin, [Ala^10,23]gurmarin, and [Ala^17,33]-gurmarin were obtained. Three analogues had native disulfide bonds, while the other six had non-native disulfide bonds. The three analogues with native disulfide bonds suppressed the response to sucrose, but not those to glucose, fructose, saccharin, or glycine in rats. In contrast, the six analogues with non-native disulfide bonds did not suppress the responses to any of these sweeteners. These results suggest that the native disulfide bonds of gurmarin are necessary for interaction with the receptor protein, and that the sucrose-specific receptor site is present in rats. © 1998 John Wiley & Sons, Inc. Biopoly 46: 65–73, 1998     

Three-dimensional structure of gurmarin,a sweet taste-suppressing polypeptide

Summary The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel -sheet. The average rmsd values between any two structures were 1.65±0.39 Å for the backbone atoms (N, C, C) and 2.95±0.27 Å for all heavy atoms. The positions of the three disulfide bridges, which could not be deterermined chemically, were estimated to be Cys³–Cys¹⁸, Cys¹⁰–Cys²³ and Cys¹⁷–Cys³³ on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in -conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in -conotoxin.Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ppm parts per million; rmsd, root-mean-square deviation - TSP 3-(trimethylsilyl)-2,2,3,3-tetradeutero-propionate     

A novel conotoxin from Conus betulinus,kappa-BtX,unique in cysteine pattern and in function as a specific BK channel modulator

A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.