Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Tryptase from rat skin: purification and properties

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.     

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.     

Synthesis and biological evaluation in vitro of selective,high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1

Human melanin-concentrating hormone (hMCH) and many of its analogues are potent but nonspecific ligands for human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R). To differentiate between the physiological functions of these receptors, selective antagonists are needed. In this study, analogues of Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), a high affinity but nonselective agonist at hMCH-1R and hMCH-2R, were prepared and tested in binding and functional assays on cells expressing these receptors. In the new analogues, 5-aminovaleric acid (Ava) was incorporated in place of the Leu(9)-Gly(10) and/or Arg(14)-Pro(15) segments of the disulfide ring. Several of these compounds turned out to be high affinity antagonists selective for hMCH-1R. Moreover, even at micromolar concentrations, they were devoid of agonist potency at both hMCH receptors and not effective as hMCH-2R antagonists. For example, peptide 14, Gva(6)- cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Ava(14,15)-Cys(16))-NH(2), (Gva = 5-guanidinovaleric acid), was a full competitive hMCH-1R antagonist (IC(50) = 14 nM, K(B) = 0.9 nM) with more than 1000-fold selectivity over hMCH-2R. Examination of various compounds with Ava in positions 9,10 and/or 14,15 revealed that the Leu(9)-Gly(10) and Arg(14)-Pro(15) segments of the disulfide ring are the principal structural elements determining hMCH-1R selectivity and ability to act as a hMCH-1R antagonist.     

Cleavage of Rabbit Myelin Basic Protein by Plasmin: Isolation and Identification of the Major Products

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.     

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.     

Synthesis and biological evaluation in vitro of a selective, high potency peptide agonist of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1

Human melanin-concentrating hormone (hMCH) is a nonselective natural ligand for the human melanin-concentrating hormone receptors: hMCH-1R and hMCH-2R. Similarly, the smaller peptide encompassing the disulfide ring and Arg(6) of hMCH, Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), Ac-hMCH(6-16)-NH(2), binds to and activates equally well both human MCH receptors present in the brain. To separate the physiological functions of hMCH-1R from those of hMCH-2R, new potent and hMCH-1R selective agonists are necessary. In the present study, analogs of Ac-hMCH(6-16)-NH(2) were prepared and tested in binding and functional assays on cells expressing the MCH receptors. In these peptides, Arg in position 6 was replaced with various d-amino acids and/or Gly in position 10 was substituted with various L-amino acids. Several of the new compounds turned out to be potent agonists at hMCH-1R with improved selectivity over hMCH-2R. For example, peptide 26 with d-Arg in place of L-Arg in position 6 and Asn in place of Gly in position 10, Ac-dArg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Asn(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), was a potent hMCH-1R agonist (IC(50) = 0.5 nm, EC(50) = 47 nm) with more than 200-fold selectivity with respect to hMCH-2R. Apparently, these structural changes in positions 6 and 10 results in peptide conformations that allow for efficient interactions with hMCH-1R but are unfavorable for molecular recognition at hMCH-2R.     

Preferred conformation, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide on the surface of neutral lipid membranes

Infrared attenuated total reflection (IR-ATR) spectroscopy and capacitance minimization (CM) were used to study the secondary structure, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide (dynorphin1-13) molecules on the surface of planar membranes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The peptide assumed a helical structure oriented perpendicularly on the membrane surface. Binding from aqueous solutions containing 10 mM KCl saturated reversibly at about a bilayer area of 110 nm2 per peptide molecule, an apparent dissociation constant of 11 microM, and rate constants of 2 X 10(2) s-1 (adsorption) and 2 X 10(-3) s-1 (desorption). The results complement those obtained by vesicle-mediated hydrophobic labeling [Gysin, B., & Schwyzer, R. (1983) Arch. Biochem. Biophys. 225, 467-474]. They indicate that the behavior of this amphiphilic peptide in contact with neutral lipid membranes may be quite different from that in molecularly disperse or micellar solutions of detergents or lysolecithins and that, in the case of dynorphin1-13, primary amphiphilicity overrules secondary amphiphilicity.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Mechanism of degradation of LH-RH and neurotensin by synaptosomal peptidases

The products of degradation of LH-RH and neurotensin by synaptosomes isolated from rat hypothalamus and cortex have been identified. LH-RH is cleaved at Tyr5-Gly6 and Pro9-Gly10 giving rise to LH-RH (1-5), LH-RH (6-10) and LH-RH (1-9). Neurotensin is cleaved at Arg8-Arg9, Pro10-Tyr11 and Ile12-Leu13, giving neurotensin (1-8), neurotensin (1-10), neurotensin (1-12) and neurotensin (9-13) as major products. While most of the peptidase activity is localized in the cytoplasmic fraction, a small but significant proportion is membrane bound. For LH-RH, the specificity of the membrane-bound activity is similar to that in the cytosol fraction; for neurotensin, the membrane fraction preferentially gives rise to the (1-10) and (1-11) peptides. The most potent inhibitors of the LH-RH and neurotensin degrading enzymes in synaptosomes are heavy metal ions (mercury and copper), p-chloromercuribenzoate and 1,10 phenanthroline.     

Estimated conformation, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide on the surface of neutral lipid membranes

Equilibrium thermodynamic and kinetic estimations were used to confirm the rather unusual conformation, orientation, and accumulation of dynorphin A-(1-13)-tridecapeptide (dynorphin1-13) on the surface of neutral lipid membranes, as observed by Erne et al. [Erne, D., Sargent, D. F., & Schwyzer, R. (1985) Biochemistry 24, 4261-4263]. I started from the premise that the most stable conformation of molecularly disperse peptides in contact with the hydrophobic phase of a membrane is helical [Henderson, R. (1979) Soc. Gen. Physiol. Ser. 33, 3-15]. Calculation of the Gibbs free energy difference for the transfer of increasing numbers m of N-terminal residues of dynorphin1-13 from their random-coil conformation in water to their alpha-helical conformation in a hydrophobic phase, with the values provided by Von Heijne and Blomberg [Von Heijne, G., & Blomberg, C. (1979) Eur. J. Biochem. 97, 175-181], showed an energy minimum at m = 9 that corresponded to the observed apparent association constant of 9 X 10(4) L/mol. This confirmed our experimental observations. The orientation of dynorphin1-13 in the interphase was estimated by calculation of the molecular amphiphilic moment A. This force vector was defined in analogy to the "helical" and "structural" hydrophobic moments of Eisenberg et al. [Eisenberg, D., Weiss, R. M., & Terwilliger, T. C. (1982) Nature (London) 299, 371-374]. It takes into account the segregation of hydrophobic and hydrophilic residues with respect to the center of the alpha-helix. A peptide located in a hydrophobic-hydrophilic gradient experiences a torque that tends to orient A in a direction perpendicular to the surfaces of equal hydrophobicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Addition of a peptide fragment on an alpha-helical depsipeptide induces alpha/3(10)-conjugated helix: synthesis, crystal structure, and CD spectra of Boc-Leu-Leu-Ala-(Leu-Leu-Lac)3-Leu-Leu-OEt

The depsipeptide Boc(1)-Leu(2)-Leu(3)-Ala(4)-Leu(5)-Leu(6)-Lac(7)-Leu(8)-Leu(9)-Lac(10)-Leu(11)-Leu(12)-Lac(13)-Leu(14)-Leu(15)-OEt(16) (1) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized from the peptide Boc-Leu-Leu-Ala-OEt (2) and a depsipeptide, Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (3). Single crystals of 1 were successfully obtained and the structure has been solved by direct methods (such as Sir2002 and Shake-and-Bake). Interestingly, 1 adopts an alpha/3(10)-conjugated helix containing a kink at the junction of peptide and depsipeptide segments, Leu3-Lac7. This is significantly different from the conformation of 3, which has a straight alpha-helical structure with standard phi and psi angles. Microcrystalline CD spectra were also studied to compare structural properties of 1 and 3. The differences between alpha/3(10)- and alpha-helices appear in these CD spectra.     

Immunohistochemical localization of dynorphin (1-8) in hypothalamic magnocellular neurons: evidence for absence of proenkephalin

Dynorphin(1-8) immunoreactivity was visualized by immunohistofluorescence in hypothalamic magnocellular neurons of the rat. No immunoreactive met-enkephalin-Arg6-Gly7-Leu8, a fragment of the adrenal medulla pro-enkephalin molecule, was detected in magnocellular neurons. However, a strong met-enkephalin-Arg6-Gly7-Leu8-like immunostaining was seen in other regions of the brain. These results suggest that in magnocellular neurons dynorphin(1-8) exists independently from pro-enkephalin and therefore the magnocellular neurons represent a third opioid peptide neuronal system in brain. These observations, however, do not rule out a coexistence of proenkephalin and dynorphin-related peptides in other regions of the brain.     

Solution structure of a peptide derived from the beta subunit of LFA-1

Cell-adhesion molecules are critical for immune response. It is well known that the inhibition of adhesion is very effective in immunotherapy and that the peptides derived from leukocyte function associated antigen (LFA-1) and intercellular adhesion molecule (ICAM-1) modulate cell-adhesion interaction. The three-dimensional structure of a cyclic peptide, Cyclo(1,12)Pen(1)-Asp(2)-Leu(3)-Ser(4)-Tyr(5)-Ser(6)-Leu(7)-Asp(8)-Asp(9)-Leu(10)-Arg(11)-Cys(12) (cLBEL) derived from the beta subunit of LFA-1 which is known to modulate homotypic T-cell-adhesion process has been studied using NMR, CD and molecular dynamics (MD) simulation. The peptide exhibits two possible conformations in solution. Structure I has a conformation with two consecutive beta-turns involving residues Tyr(5)-Ser(6)-Leu(7)-Asp(8) and Asp(9)-Leu(10)-Arg(11)-Cys(12). Structure II has a beta-turn at Tyr(5)-Ser(6)-Leu(7)-Asp(8) and forms a beta-hairpin type of conformation.     

Characterization of the C-terminal flanking peptide of human beta-preprotachykinin

The nucleotide sequence of cDNA encoding the common biosynthetic precursor of substance P, neurokinin A and neuropeptide K (beta-preprotachykinin) predicts that, in the human, the precursor contains a C-terminal flanking peptide of 19 amino acid residues [beta-preprotachykinin(111-129)-peptide]. Using an antiserum raised against synthetic human beta-preprotachykinin(117-126)-peptide in radioimmunoassay, we have demonstrated that an extract of a human neuroendocrine tumor of the adrenal medulla contained approximately equimolar concentrations of C-terminal preprotachykinin immunoreactivity (C-PPT-IR), substance P and neurokinin A. The C-terminal preprotachykinin flanking peptide was purified to homogeneity and its primary structure was determined. The amino acid sequence of the peptide, Ala-Leu-Asn-Ser-Val-Ala-Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, indicates identity with beta-preprotachykinin(111-126)-peptide. The data suggest that the C-terminal flanking peptide, like the tachykinins, is packed into secretory storage vesicles but the Arg127-Arg128-Arg129 residues in human beta-preprotachykinin are removed from the peptide by the action of endogenous processing enzyme(s).     

High-resolution NMR studies of fibrinogen-like peptides in solution: resonance assignments and conformational analysis of residues 1-23 of the A alpha chain of human fibrinogen

The proton resonances of the following synthetic linear human fibrinogen-like peptides were completely assigned with two-dimensional NMR techniques in solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Gly(12)-Gly(13)-Gly(14)- Val(15)-Arg(16)-Gly-Pro-Arg-Val-Val-Glu-Arg (F10), Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly(13)-Gly(14)-Val-Arg (F11), and Gly-Pro-Arg-Val-Val-Glu-Arg (F12). No predominant structure was found in the chain segment from Ala(1) to Gly(6) for F10 in both H2O and dimethyl sulfoxide. The previous suggestion that there is a hairpin loop involving residues Gly(12) to Val(15) in the A alpha chain of human fibrinogen is supported by the slow backbone NH exchange rates of Gly(14) and Val(15), by an unusually small NH chemical shift of Val(15), and by strong sequential NOE's involving this region in F10. This local chain fold within residues Asp(7) to Val(20) may place the distant Phe residue near the Arg(16)-Gly(17) peptide bond which is cleaved by thrombin.     

Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity

A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, "leumorphin") to the tridecapeptide dynorphin B (Dyn B, "rimorphin"). This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent Km for the substrate of 0.58 microM. Surprisingly, a synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent Ki of 3.7 microM. The converting activity was also inhibited by Dyn A-(6-17) but not by Dyn A-(8-17), suggesting a role of Arg6-Arg7 in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition. Beta-Endorphin was an effective inhibitor of converting activity, and [alpha-N-acetyl]beta-endorphin was not, indicating a crucial role of the free NH2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent Ki of 39 nM. The converting activity was also inhibited substantially by ACTH-(1-13) but not by alpha-MSH, again indicating a requirement of the free NH2-terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Study of bradykinin conformation in a dimethylsulfoxide solution by two-dimensional 1H-NMR spectroscopy

The role of charged groups of the nonapeptide bradykinin in stabilization of its spatial structure in dimethyl sulfoxide solution was investigated. The signal assignment in the 1H-NMR spectra was achieved by means of two dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The changes in the NH and C alpha H proton chemical shifts of the Arg1 and Arg9 residues, variations both in temperature coefficients of chemical shifts of NH-resonances and coupling constants, as well as the appearance of additional NOE cross-peaks in NOESY spectra for d alpha N and d beta N 1H-1H distances were revealed by comparing the NMR spectra for two states--with the protonated C-terminal carboxyl group and deprotonated one. The experimental results are in agreement with the assumption that the conformation of the peptide in (CD3)2SO is stabilized by electrostatic interaction between the oppositely charged N- and C-terminal groups. The conformation with deprotonated alpha-carboxyl group is characterized by two beta-turns in the sequences Pro2-Pro-Gly-Phe5 and Ser6-Pro-Phe-Arg9.     

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.