Prolonged tissue damage or injury often leads to chronic pain states such that noxious stimuli evoke hyperalgesia and innocuous tactile stimuli evoke pain (allodynia). The neuropeptide nociceptin, also known as orphanin FQ, is an endogenous ligand for the orphan opioid-like receptor which induces both hyperalgesia and allodynia when administered by injection through the theca of the spinal cord into the subarachnoid space (that is, intrathecally). Here we show that the nociceptin precursor contains another biologically active peptide which we call nocistatin. Nocistatin blocks nociceptin-induced allodynia and hyperalgesia, and attenuates pain evoked by prostaglandin E2. It is the carboxy-terminal hexapeptide of nocistatin (Glu-Gln-Lys-Gln-Leu-Gln), which is conserved in bovine, human and murine species, that possesses allodynia-blocking activity. We have also isolated endogenous nocistatin from bovine brain. Furthermore, intrathecal pretreatment with anti-nocistatin antibody decreases the threshold for nociceptin-induced allodynia. Although nocistatin does not bind to the nociceptin receptor, it binds to the membrane of mouse brain and of spinal cord with high affinity. Our results show that nocistatin is a new biologically active peptide produced from the same precursor as nociceptin and indicate that these two peptides may play opposite roles in pain transmission.

Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.

X-ray analysis has been carried out on a crystal of the collagen model peptide (Hyp(R)-Hyp(R)-Gly)10 [where Hyp(R) is 4(R)-hydroxyproline] with 1.5 A resolution. The triple-helical structure of (Hyp(R)-Hyp(R)-Gly)10 has the same helical parameters and Rich and Crick II hydrogen bond patterns as those of other collagen model peptides. However, our full-length crystal structure revealed that almost all consecutive Hyp(R) residues take the up-up pucker in contrast to putative down-up puckering propensities of other collagen model peptides. The unique feature of thermodynamic parameters associated with the conformational transition of this peptide from triple helix to single coil is that both enthalpy and entropy changes of the transition are much smaller than those of other model peptides such as (Pro-Pro-Gly)10 and (Pro-Hyp(R)-Gly)10. To corroborate the precise structural information including main- and side-chain dihedral angles and intra- and interwater bridge networks, we estimated the degrees of hydration by comparing molecular volumes observed experimentally in solution to those calculated ones from the crystal structure. The results showed that the degree of hydration of (Hyp(R)-Hyp(R)-Gly)10 is comparable to that of (Pro-Hyp(R)-Gly)10 in the triple-helical state, but the former was more highly hydrated than (Pro-Hyp(R)-Gly)10 in the single-coil state. Because hydration reduces the enthalpy due to the formation of a hydrogen bond with a water molecule and diminishes the entropy due to the restriction of water molecules surrounding a peptide molecule, we concluded that the high thermal stability of (Hyp(R)-Hyp(R)-Gly)10 is able to be described by its high hydration in the single-coil state.

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.

β‐Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β‐linked glucans are constantly exposed to degradation by various endo‐β‐glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo‐β‐glucanases inhibitors, such as xyloglucan‐specific endo‐β‐1,4‐glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three‐dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo‐222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S. Database 
Coordinates and structure factors have been deposited in the Protein Data Bank Japan (PDBj) (http://www.pdbj.org/) under the accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3AUP. Structured digital abstract  http://www.uniprot.org/uniprot/P13917  http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/P13917 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8151069) http://www.uniprot.org/uniprot/P13917  http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/P13917 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0028 (View Interaction http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8151079, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8151085) http://www.uniprot.org/uniprot/P13917  http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/P13917 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071 (View Interaction http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8151100, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8151094)

To determine the mode of disulphide bond formation in conotoxin GI, a tridecapeptide amide with 4 Cys residues, all 3 of its peptides having different modes of disulphide‐bond formation were synthesized by solution procedure using selectively removable protective groups at the Cys residues. After deprotection with HF, one pair of acetamidomethyl groups was left unremoved, and then two sets of disulphide bonds were formed selectively. The toxic potency in mice of one product was comparable with that reported for native conotoxin GI and was almost 10‐fold as high as that of the other two products. The toxicity of the native toxin reportedly is not regenerated upon reduction and reoxidation, but this study showed that the most toxic product was the most readily formed one.

Previously, we isolated a 4-kDa peptide capable of binding to a 43-kDa receptor-like protein and stimulating protein kinase activity of the 43-kDa protein in soybean. Both of them were found to localize in the plasma membranes and cell walls. Here, we report the physiological effects of 4-kDa peptide expressed transiently in the cultured carrot and bird's-foot trefoil cells transfected with pBI 121 plasmid containing the 4-kDa peptide gene. At early developmental stage, the transgenic callus grew rapidly compared to the wild callus in both species. Cell proliferation of in vitro cultured nonembryogenic carrot callus was apparently affected with the 4-kDa peptide in the medium. Complementary DNAs encoding the 4-kDa peptide from mung bean and azuki bean were cloned by PCR and sequenced. The amino-acid sequences deduced from the nucleotide sequences are homologous among legume species, particularly, the sites of cysteine residues are highly conserved. This conserved sequence reflects the importance of intradisulfide bonds required for the 4-kDa peptide to perform its function. Three dimensional structure of the 4-kDa peptide determined by NMR spectroscopy suggests that this peptide is a T-knot scaffold containing three b-strands, and the specific binding activity to the 43-kDa protein and stimulatory effect on the protein phosphorylation could be attributed to the spatial arrangements of hydrophobic residues at the solvent-exposed surface of two-stranded b-sheet of 4-kDa peptide. The importance of these residues for the 4-kDa peptide to bind to the 43-kDa protein was indicated by site-directed mutagenesis. These results suggest that the 4-kDa peptide is a hormone-like peptide and the 43-kDa protein is involved in cellular signal transduction of the peptide.Keywords: hormone-like peptide; plants; NMR; threedimensional structure; site-directed mutagenesis; physiological function.A 43-kDa protein found in the soybean seeds is a glycoprotein with sedimentation coefficient of 7S and isoelectric point ranging from 9.05 to 9.26 [1]. This protein has been classified into the category of globulin, which is soluble only in high ionic strength of salt solutions [1]. It consists of a and b subunits linked by disulfide bridge(s). There are a cysteine-rich domain in the N-terminal side of a subunit, a putative transmembrane domain in the b subunit [2], and a consensus sequence of ATP-binding site indispensable for protein phosphorylation activity [2]. The 43-kDa protein has autophosphorylation activity and protein kinase activity about two thirds of tyrosine kinase activity of the rat insulin receptor [3]. Immunocytochemistry has indicated that the 43-kDa protein is localized in the plasma membranes and the middle lamellae of cell walls [4], suggesting that it is a receptor-like protein. Western blotting and DNA cloning experiments revealed that these proteins are structurally similar to the 43-kDa protein and distribute in a number of legume species such as azuki bean, cowpea, French bean, lupin, mung bean and winged bean [5,6]...

Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol-disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria.