Specific cleavage of histidine-containing peptides by copper (II)

Copper (11) cleaves with moderate specificity peptides containing Ser-His or Thr-His sequences, at the N-terminal side of the hydroxyaminoacyl residue. The reaction is slow, and is first-order in peptide: Cu^II complex, with a half-life of several hours at 62 ° C in sodium bicarbonate buffer, pH 8. Cleavage of other histidine-containing peptides also occurs, at a rate around 10–100-fold less. EDTA completely quenches the cleavage. The reaction is stoichiometric in Cu^II and is inhibited by amine-containing buffer components; Tris at 19 MM inhibits cleavage by 50%. The reaction has a complex pH-dependence, being very slow below pH 5, and with rates increasing with pH from pH 7 to pH 9.5. Slower degradative side reactions do occur, with destruction of tyrosine residues, particularly in the presence of high concentrations of chloride ion, but the specific cleavage appears to be a hydrolysis, as determined by amino-acid analysis and mass spectrometry of the products. The cleavage is clearly different from the previously described oxidative degradation of proteins catalysed by copper ions. Cleavage of denatured IgG protein occurs with sufficient specificity to reveal distinct bands on SDS-polyacrylamide gel electrophoresis under reducing conditions. © Munksgaard 1996.     

Selective isopeptide bond formation: coupling ubiquitin67–76 with histone 2A114–128 by use of the transfer active ester condensation technique

Abstract: A peptide, ubiquitin^67–76-histone 2A^114–128 fragment (UBH2AF), was synthesized by selective formation of an isopeptide bond between the C-terminus of ubiquitin^67–76 and the ε-amino group of lysine-119 in histone 2A^114–128 which contained 4 lysine residues at positions 118, 119, 125 and 127, respectively. The transfer active ester condensation technique, together with the Tnm amine protecting group, were used successfully in the peptide segment coupling reaction. Leu⁶⁷-His-Leu-Val-Leu-Arg-Leu-Arg-Gly- Gly ⁷⁶ | Val¹¹⁴-Leu-Leu-Pro-Lys(NH₂)- Lys¹¹⁹ -Thr-Glu-Ser-His-His-Lys(NH₂)-Ala-Lys(NH₂)-Gly¹²⁸ UBH2AF     

Activity of VIP,somatostatin and other peptides in the mouse vas deferens assay

Non-opiate peptides such as vasoactive intestinal peptide (VIP) and somatostatin were tested for their effects on electrically induced contractions of the vas deferens. VIP (ED₅₀=2.7×10^?8M) and to a lesser extent somatostatin (ED₅₀=5.2×10^?8M) were found to be in the same general range of activity as enkephalin and the endorphins in this system. Human pancreatic polypeptide (HPP) exerted a biphasic effect, inhibiting the contractions at high concentrations but enhancing them at lower concentrations. A number of other naturally occurring brain peptides were ineffective at concentrations of 1×10^?6 M. Several somatostatin analogues were tested and their activity on the vas deferens was found to more closely parallel their potency to inhibit the release of gastric acid than of growth hormone. In contrast to the brain opiates, however, the inhibitory effects of VIP, somatostatin and its analogues, and HPP were not reversed by the opiate antagonist naloxone. The results suggest that the vas deferens can be readily used for evaluation of analogues of VIP, somatostatin, and other peptides.     

Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205–316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu³⁰² residue into fragments 205–302 and 303–316. Upon incubation for 2–5 days of fragment 205–302 with a 5–fold excess of peptide 303–316, prepared by solid phase synthesis, with V8-protease in 0.1M ammonium acetate, pH6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to ñ90% (based on fragment 205–302). The same procedure was used to prepare also the thermolysin fragments 205–315 and 205–311 by enzymatic coupling of fragment 205–302 to peptide 303–315 or 303–311, these last prepared by proteolytic digestion of the synthetic peptide 303–316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205–316 of thermolysin at the level of its helical segment encompassing residues 301–312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Conformation and inhibitory properties of peptides based on the tissue kallikrein-aprotinin complex

A series of peptides encompassing the primary binding segment (residues 12–19) of aprotinin has been synthesized and tested for their ability to inhibit procine pancreatic kallikrein. A minimum sequence of five amino acids spanning residues 12-16 of aprotinin is necessary for inhibition of porcine pancreatic kallikrein. An octapeptide homologous with the binding segment of aprotinin has a K_ivalue of 1.2 × 10^?4M. The solution structure of the octapeptide was studied by one- and two-dimensional NMR methods for comparison with the known structure of the segment of aprotinin that contacts tissue kallikrein. NMR experiments suggest that the peptide is either a random coil or that it samples several conformations on the NMR time scale. Analysis of the molecular dynamics trajectory of the octapeptide also suggests that the peptide is highly flexible. Thus, inhibition by the octapeptide occurs because of its homology with residues 12-19 of aprotinin. Moreover, the absence of a stable solution conformation similar to that of the binding segment of aprotinin is consistent with the 150 000-fold increase in K_i of the octapeptide compared to intact aprotinin.     

Structural comparison of alanine-substituted analogues of the calcitonin gene-related peptide 8-37 Importance of the C-terminal segment for antagonistic activity

Replacement of specific residues of the antagonistic fragment human calcitonin gene-related peptide 8–37 (hCGRP 8–37) by alanine residues produces good antagonists to CGRP1 receptors when the replacement is made at positions 17 and 20 but a poor antagonist when the replacement is made at position 21. The solution structures of hCGRP 8-37 and of the three alanine analogues have been determined by two-dimensional ¹H NMR spectroscopy and molecular modeling. Following the complete assignment of the NMR spectra, a comparison of the chemical shifts and of the temperature dependence of the amide chemical shifts showed that these parameters differed for [Ala¹⁷]-hCGRP 8–37 and [Ala²⁰]-hCGRP 8–37 relative to hCGRP 8–37 in the N-terminal and central segments but not in the C-terminal segment (residues 31–37). In the case of [Ala²¹]-hCGRP 8–37, differences were observed all along the chain. Molecular modeling calculations were performed by distance geometry, simulated annealing and energy minimization using NOE distance constraints. Molecular models showed a structural homology between [Ala¹⁷]-hCGRP 8–37, [Ala²⁰]-hCGRP 8–37 and hCGRP 8–37 in the C-terminal segment Asn³¹-Phe³⁷ as well as hydrogen bonding between Val²⁸ and Asn³¹. These structural similarities are not observed with [Ala²¹]-hCGRP 8–37. Therefore, the structure of the C-terminal segment of hCGRP 8–37 appears to be critical for antagonistic activity at CGRP1 receptors.     

Isolation,purification and 13C-and 1H-n.m.r. assignments of peptide [1–24] of human serum albumin

Isolation, purification and 360 MHz ¹H- and ¹³C-n.m.r. spectra of the residue corresponding to the NH₂-terminal peptide fragment [1–24] of human serum albumin are reported. The various resonances have been assigned to individual amino acid residues and their spatial microenvironment has been determined in a straightforward manner on the basis of (i) pH dependent chemical shifts; (ii) combined use of multiple and selective proton-decoupled ¹H- and ¹³C-n.m.r. spectra; (iii) the characteristic pK values exhibited by protons adjacent to sites of ionization in the molecule; and (iv) comparison of the spectra with the NH₂-terminal tripeptide segment of human albumin. The pK values of different ionizable groups all fall in the normal range expected for each titrating sites and support a model of peptide fragment [1–24] in which there is no special structure-forming strong associations. These results are in agreement with those obtained by CD spectroscopy.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

SYNTHESIS OF AN OPEN-CHAIN ASYMMETRICAL CYSTINE PEPTIDE CORRESPONDING TO THE SEQUENCE A18–21 -B19–26 OF BOVINE INSULIN BY SOLID PHASE FRAGMENT CONDENSATION

An insulin fragment containing residues A^18–21 and B^19–26 linked by the disulfide bond between residues A²⁰ and B¹⁹ was synthesized. The sequence B^21–26 was assembled on a solid support by the Merrifield technique. The protected fragments A^18–21 and B^19–20 were prepared by conventional methods. After forming the disulfide bridge through cleavage of the S-thiocarbonate derivative of A^18–21 by the thiol peptide B^19–20, the resulting asymmetrical cystine peptide A^18–21—B^19–20 was coupled via the carboxyl group of residue B²⁰ to the free NH₂-terminal amino group of the protected B^21–26 resin. The product was deprotected, cleaved from the resin, and purified to give the homogeneous dodecapeptide A^18–21-B^19–26.     

Crystal structure of L-lysyl-L-glutamic acid dihydrate

L-Lysyl-L-glutamic acid dihydrate, C₁₁N₃O₅H₂₁·2H₂O, crystallizes in the monoclinic space group P2₁ with a = 12.474(2), b = 5.020(1), c = 13.157(2) Å, β= 114.69(1)° and Z = 2. The crystal structure was solved by direct methods and refined to an R value of 0.037 using full matrix least-squares method. The molecule exists as a double zwitterion with both the amino and carboxyl groups ionised. The peptide has a folded conformation with its Lys residue trans and Glu residue gauche^?gauche⁺. The side chains of the Lys and Glu residues correspond to all trans and folded (g^?g^?g^?) conformations respectively. The terminal carboxyl group forms hydrogen bonds with the ξ-amino group of the lysine side chain. The head-to-tail interaction often seen in peptide crystals is absent in the present structure. In the extended crystal structure water molecules form channels along the b direction and are enclosed within helically arranged hydrogen bonds formed by the lysine side chain and the peptide backbone.     

Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-C～S⁶,D-Cys¹¹,Lys¹²,Nle¹⁷,Val²⁶,Thr²⁸]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys¹²,Cys¹⁷,Val²⁶,Cys²⁸]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (>40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor–active conformation of VIP.     

Trypsin-mediated semisynthesis of salmon calcitonin

Salmon calcitonin, CT(1-32)·NH₂, was synthesised by the trypsin-mediated coupling of the peptide fragments CT(1-24) and CT(25-32)·NH₂, prepared by conventional Fmoc solid-phase chemistry. Optimal conditions regarding reaction time course, pH, proportion of catalyst, substrate concentration and composition of the reaction medium were determined from initial studies on the coupling of CT(1-11) to CT(12-24) and of CT(12-24) to CT(25-32)·NH₂. For the final successful semisynthesis, we found that it was unnecessary to protect lysine residues not involved in the coupling, and that secondary hydrolysis at these sites could be prevented by increasing the pH of the reaction medium. The reaction achieved equilibrium after 30-45 min, with overall conversion of around 30% of the initial amount of CT(1-24) substrate into product. Yields were depressed due to cyclisation of the CT(1-24) substrate via air-oxidation of the Cys¹ and Cys⁷ residues.     

Solution structure of the phosphorylated sites of ribosomal protein S6 by 1H NMR spectroscopy

An increase in the rate of protein synthesis is found to be accompanied by phosphorylation of the 40S ribosomal protein S6. Treatment of S6 by cyanogen bromide produced three fragments, and one of the fragments of S6, which is a C-terminal portion of S6 (M_r? 4000), contains all phosphorylation sites of S6. The C-terminal fragment of S6 contains seven serines. S6 kinase phosphorylates S6 specifically, i.e. five serines in the C-terminal of S6 are phosphorylated. The three-dimensional structure of S6 peptide was studied in 50% trifluoroethanol/50% H₂O solution by ¹H NMR with combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it takes an α-helix between Glu5 and Arg21 and a distorted helical structure for the following three residues, but no rigid structure was present from Ser25 through the C-terminus and for the N-terminal region (Lys1-Lys4). The specificity of the phosphorylation of the peptide is discussed from a structural aspect. © Munksgaard 1996.     

Angiotensin II binding to zona glomerulosa cells from rabbit adrenal glands.

Tritiated angiotensin II binds in a highly specific manner to zona glomerulosa cells prepared from the adrenal cortex of male rabbits. The reaction is a time-dependent process which obeys second-order kinetics (k₁ = 2.4 × 10⁵ M^?1 sec^?1) and reaches saturation in 5–7 min. Dissociation of the angiotensin II-cell complex is rapid (t_{1 2} = 100 sec) and obeys first-order kinetics for the first 3 min (k_?1 = 6.9 × 10^?3 sec^?1).Increased binding was observed with NaCl concentrations from 0 to 40 × 10^?3 M; however, concentrations from 40 to 140 × 10^?3 M decreased binding. Neither MgCl₂ nor Cll₂ at concentrations of 0 to 4 × 10^?3 M alter the binding of angiotensin II to zona glomerulosa cells. A significant decrease in binding was observed with increasing concentrations of KCl (0 to 140 × 10^?3 M).Temperature studies indicate that initially binding is more rapid at 37° (37° > 25° > 0°). However, binding of angiotensin II decreases after 3 min at 37° and after 7 min at 25°. Binding at 0° did not reach a plateau in the 15-min period studied.     

Nitroxyl-induced T1 relaxation rates as a probe of conformation in peptide hormones

Proton spin-lattice relaxation rates in the N-H region of the n.m.r. spectra of aqueous LHRH and angiotensin II were measured in the presence of varying concentrations of 2,2,6,6-tetramethylpiperidinoxyl. At peptide concentrations of 3–7 mM and nitroxyl concentrations up to three times the peptide concentration, the relaxation rate is linearly dependent on nitroxyl concentration. Second order rate constants for nitroxyl induced relaxation in water are in the 200–1000s^-1 M^-1 range, and are dependent more on conformational factors than on side chain bulk. Exclusion of the radical from the hydration sphere of imidazolium ion appears to occur. The measurements for LHRH agree with earlier observations of the effect of the nitroxyl on linewidths. A two- to threefold decrease in sensitivity of the amide protons of at least three of the residues in angiotensin II when the His⁶ imidazole is protonated indicates a conformational transition related to this ionization.     

Conformational dynamics and kinetics of peptide antagonist interactions with interleukin-1 receptor. Fluorescence studies using the NBD-labelled peptide AF12415

Interleukin-1 plays a key role in the inflammatory response provoked by various disease states and inhibition of its action can bring therapeutic benefits. Steady-state and time-resolved studies of the intrinsic tryptophan fluorescence of the free soluble Type I form of interleukin-1 receptor (IL-1R) reveal that the rotational motions of the three major domains are strongly associated. Bound peptide antagonists are buried in hydrophobic regions, but a flexible association permits access to species from the aqueous phase. Ligand binding does not lead to rigidification of the receptor structure. The kinetics and mechanism of complex formation and dissociation, involving IL-1R with receptor antagonist protein (IL-1ra) and with peptides AF11733 (15 aa) and AF10961 (21 aa) were determined with the aid of peptide AF12415 (15 aa) labeled at its N-terminus by the NBD fluorophore, which exhibits a five-fold increase in emission intensity at 540 nm on binding of the peptide to IL-1R. The magnitude of the ON rate constant, typically 1 × 10⁶ M^?1 s^?1, implies the existence of an intermediate ‘encounter complex’ involving interactions of low specificity. Readjustments of the initial encounter complex leads to a final complex where very specific interactions dominate. The first-order rate constant for this latter process is the most sensitive indicator of the true peptide affinity for the receptor binding site, and thus provides a better criterion than the apparent OFF rate (typically 2 × 10^?3 s^?1) for discrimination of peptide antagonists. © Munksgaard 1997.     

Tritiated D-ala1-peptide T binding: A pharmacologic basis for the design of drugs which inhibit HIV receptor binding

The HIV virus initiates its infectious cycle through a high-affinity binding interaction between the envelope protein gp 120 and its receptor, the T4 (or CD4) molecule. An octapeptide sequence, termed peptide T, present in the second variable region of gp 120 from the ARV isolate, has been implicated as the attachement site. The core peptide required for activity has been further refined to a pentapeptide, and homologous pentapeptides are similarly positioned in 21 other sequenced HIV isolates. Utilizing a novel direct binding assay of ³H-D-ala¹-peptide T, we now report that synthetic peptides derived from these other isolates are potent competitors of peptide T binding to T4-positive lymphocytes and brain membranes. Direct peptide T binding is also competable by purified virion gp 120, indicating that these ligands are interactive at the same receptor. Peptide T has sequence relatedness to the peptide vasoactive intestinal peptide (VIP), and VIP and its relevant homologous pentapeptide, VIP[7–11], are also potent inhibitors of peptide T binding. To determine the essential structural features responsible for receptor activity we have studied a series of synthetic peptides substituted with single D-amino acid residues. These data reveal that the tyrosine of position 7 in peptide T, present in all natural viral isolates, is obligate for receptor activity. Structure/function analysis for a large number of analogs is presented. Significantly, this binding assay is highly correlated with peptide bioactivity in several independent systems, indicating that this methodology can be used for rapid screening of novel, potential anti-AIDS therapeutics whose target is inhibition of virus gpl20 binding.     

Differential Effects of Nicorandil on the Vasodepressor Responses to Vasoactive Polypeptides Administered Intravenously to Rats

The effects of nicorandil on vasodepressor responses to vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and substance P have been examined in anaesthetized rats. Intravenous bolus injections of VIP (0.3 μg kg^?1), CGRP (0.1 μg kg^?1) and substance P (0.1 μg kg^?1) induced reductions of blood pressure accompanied by slight increases (less than 5%) in heart rate. Nicorandil infused intravenously at 10 or 30 μg kg^?1 min^?1 significantly augmented the vasodepressor responses to VIP and CGRP but did not modify the responses to substance P and acetylcholine (0.1 μg kg^?1). Intravenous treatment with glibenclamide (20 μg kg^?1) significantly attenuated not only the vasodepression caused by VIP and CGRP, but also the enhancement of the effects of the agents by nicorandil. These results indicate that nicorandil can enhance the action of VIP and CGRP, in rats, at least partly through ATP-sensitive K⁺-channel activation.     

Solvent-free K–CaO/Ca(OH)2 mixed-phase nanocatalytic single-step methanolysis,ethanolysis and aminolysis of Pongamia pinnata triglycerides

In this report, the K–CaO/Ca(OH)₂ mixed-phase nanodendrites were prepared and used as a heterogeneous catalyst for the one-pot solvent-free synthesis of methyl esters, ethyl esters and alkyl amides from Pongamia pinnata triglycerides. A modified incipient-wetness chemical impregnation method was used to prepared potassium hydroxide doped CaO, MgO, and ZnO nanocrystallites. The prepared K–CaO/Ca(OH)₂ nanocrystallites were found as the most efficient heterogeneous catalyst (4%, w/w) for aminolysis (6:1 M ratio of diethanolamine/oil, 110 °C), methanolysis (9:1 M ratio of methanol/oil at 65 °C) and ethanolysis (9:1 M ratio of ethanol/oil at 75 °C) of Pongamia pinnata oil and took 40 min for the completion (99% yield) of all the reactions. The K–CaO/Ca(OH)₂ nanodendrites were found to be efficient at room temperature (25–30 °C) also and reused for 10 reaction cycles. The pseudo-first-order reaction kinetic model was applied and the first-order rate constant was calculated as 0.112 min^?1, 0.093 min^?1, and 0.118 min^?1 for the methanolysis, ethanolysis, and aminolysis of Pongamia pinnata oil, respectively.     

H n.m.r. spectrum from the flexible N-terminal segment of Streptomyces subtilisin inhibitor

The ¹H n.m.r. spectrum of Streptomyces subtilisin inhibitor shows a limited number of unusually sharp signals at room temperature. Some of these signals are assigned uniquely to protons of the side chains of the N-terminal segment, Aspl-Ala2-Pro3-Ser4-Ala5-Leu6-Tyr7-based on experiments of spin decoupling, pH titration, and enzymatic cleavage of the protein. Quantitative examination of these signals indicates that the N-terminal end of this protein is heterogeneous in that the protein contains a considerable fraction whose sequence starts with Ala2 rather than with Asp1. The pK_a values for the amino groups of Asp1 and Ala2 exposed at the N-terminus are determined to be 8.9 ± 0.4 and 9.0 ± 0.1, respectively. Furthermore, examination of the line-widths of the methyl proton resonances of Ala2 and Ala5 residues indicates that the N-terminal peptide segment is free and undergoes rapid segmental motions in the order of 10^?9s.