Biological and physical properties of a beta-endorphin analog containing only D-amino acids in the amphiphilic helical segment 13-31

Our approach to the modeling of beta-endorphin has been based on the proposal that three basic structural units can be distinguished in the natural peptide hormone: a highly specific opiate recognition sequence at the N terminus (residues 1-5) connected via a hydrophilic link (residues 6-12) to a potential amphiphilic helix in the C-terminal residues 13-31. Our previous studies showed the validity of this approach and have demonstrated the importance of the amphiphilic helical structure in the C terminus of beta-endorphin. The present model, peptide 5, has been designed in order to evaluate further the requirements of the amphiphilic secondary structure as well as to determine the importance of this basic structural element as compared to more specific structural features which might occur in the C-terminal segment. For these reasons, peptide 5 retains the three structural units previously postulated for beta-endorphin; the major difference with regard to previous models is that the whole C-terminal segment, residues 13-31, has been built using only D-amino acids. In aqueous buffered solutions as well as in 2,2,2-trifluoroethanol-containing solutions, the CD spectra of peptide 5 show the presence of a considerable amount of left-handed helical structure. Enzymatic degradation studies employing rat brain homogenate indicate that peptide 5 is stable in this milieu. In delta- and mu-opiate receptor-binding assays, peptide 5 shows a slightly higher affinity than beta-endorphin for both receptors while retaining the same delta/mu selectivity. In opiate assays on the guinea pig ileum, the potency of peptide 5 is twice that of beta-endorphin. In the rat vas deferens assay, which is very specific for beta-endorphin, peptide 5 displays mixed agonist-antagonist activity. Most remarkably, peptide 5 displays a potent opiate analgesic effect when injected intracerebroventricularly into mice. At equal doses, the analgesic effect of peptide 5 is less than that of beta-endorphin (10-15%) but longer lasting. In conjunction with our previous model studies, these results clearly demonstrate that the amphiphilic helical structure in the C terminus of beta-endorphin is of predominant importance with regard to activity in rat vas deferens and analgesic assays. The similarity between the in vitro and in vivo opiate activities of beta-endorphin and peptide 5, when compared to the drastic change in chirality in the latter model, demonstrates that even a left-handed amphiphilic helix formed by D-amino acids can function satisfactorily as a structural unit in a beta-endorphin-like peptide.     

beta-Endorphin: analgesic and receptor binding activity of non-mammalian homologs

Analgesic potencies of turkey, ostrich and des-acetyl salmon beta-endorphins have been measured in the tail-flick test and binding affinities determined by radio-receptor assay. The duration of analgesia and the slope of the dose-response curves generated by these peptides are similar to those elicited by mammalian beta-endorphins. This suggests that they act in vivo and in vitro on the same population of opiate receptors. The ratio of binding to analgesic potencies observed for these peptides varies nearly sixfold. Structure-activity analysis suggests that a basic side-chain at position 9 is required in order to produce a high opiate activity both in vivo and in vitro. A reexamination of the biological activities of camel beta-endorphin shows that the analgesic potency and binding affinity of this peptide are respectively 171 and 2.7 times higher than human beta-endorphin. His-27 and/or Gln-31 may contribute to this increased potency. The dissociation of radioreceptor binding affinity from analgesic potency in these naturally occurring beta-endorphin homologs suggests that either the conditions under which the binding assay is performed mask the true binding potency in the brain or that, once bound to the appropriate receptor, these homologs do not possess equal ability to produce biological effects.     

Opiate binding properties of naturally occurring N- and C-terminus modified beta-endorphins

Beta-endorphin is further processed within the pituitary and brain by either N-terminal acetylation, carboxy-terminal proteolysis, or both. These naturally occurring analogues are stored intracellularly and, in some tissues, represent the majority of beta-endorphin immunoreactivity detected by antisera. It is therefore critical to determine their relative potencies at the opiate receptor. This study demonstrates that cleavage of the C-terminus tetrapeptide brings about a 10-fold decrease in opiate binding potency of either camel or human beta-endorphin. N-Acetylation, on the other hand, causes over a thousand fold loss in opiate potency rendering the peptide effectively inactive. Since unmodified beta-endorphin is approximately equipotent at multiple opiate receptors, we tested for possible differential shifts towards mu or delta-type receptors which may result from the modification. Our results show no change in selectivity, but simply an overall loss of potency.     

Inhibition of analgesia by C-terminal deletion analogs of human beta-endorphin

Human beta-endorphin (beta h-EP) analogs of variable chain lengths have been investigated for their potency in inhibiting analgesia induced by beta h-EP or by the potent opiate etorphine. It was found that beta h-EP-(1-28) inhibits the analgesic effect of beta h-EP and etorphine when co-injected intracerebroventricularly into mice. Antagonism by competition at same opioid receptor subtypes is suggested from parallel shifts of the dose-response curve of etorphine or beta h-EP in the presence of increasing doses of beta h-EP-(1-28). On a molar basis, beta h-EP-(1-28) is nearly 10 times more potent than naloxone. The reduction of the chain length from residues 1-28 to 1-27 lowered the antagonist potency while further reduction of the peptide chain led to a complete loss of inhibitory activity. From comparison of the opioid-receptor binding affinity, analgesic activity and antagonist potency, it is concluded that the C-terminus of beta-EP is critical to the biological efficacy of the molecule and that the antagonist activity of C-terminal deletion analogs is probably mediated through residues 27 and 28.     

Synthesis and biological activity of a lysine-containing cyclic analog of [Leu5]enkephalin

The cyclic analogue of [Leu5]enkephalin--cyclo (Lys-Tyr-Gly-Gly-Phe-Leu) and two corresponding linear hexapeptides with lysine residue attached to the N- or C-terminus of the molecule have been synthesized by classical methods of peptide chemistry. The addition of lysine residue to the N-terminus of cyclization of the molecule reduce the interaction of these analogues with both central and peripheral opiate receptors. The addition of lysine residue to the C-terminus of the molecule through the epsilon-amino group does not affect the interaction of the analogue with mu-receptors but reduces approximately tenfold its affinity for delta-receptors. All three analogues have analgesic potency similar to that of [Leu5]enkephalin as assayed after intracisternal administration to mice.     

Synthesis and receptor binding activity of elephant beta-endorphin, a beta-endorphin homolog with highly potent analgesic activity.

Elephant beta-endorphin and its analog, elephant beta-endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant beta-endorphin was five to six times more potent than human beta-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16, 33-37), tail flick test for intracerebroventricularly administered beta-endorphin showed that the antinociceptive potency of elephant beta-endorphin was seven to eight times higher than that of human beta-endorphin in mice. Results from both studies suggest that elephant beta-endorphin was a much more potent antinociceptive agent than human beta-endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.     

Examination of the requirement for an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides

In our approach to beta-endorphin modeling, we have proposed that the biological properties of the natural peptide are determined by the combination of three basic structural units: a highly specific opiate recognition sequence at the NH2 terminus (residues 1-5) connected via a hydrophilic peptide link (residues 6-12) to a potential amphiphilic helix in the COOH-terminal residues 13-31. In the alpha-helical conformation the hydrophobic domain twists around the length of the helix and covers almost one-half of its surface. The other distinctive features of the helix include its basicity and the two aromatic residues Phe18 and Tyr27. In contrast to previous models we have studied, peptide 4 is a "negative" model in the sense that it was designed and examined in order to determine how the lack of a well defined amphiphilic structure affects the biological properties of beta-endorphin. For this purpose, peptide 4 retains the three structural units previously postulated for beta-endorphin, but the amino acids of the 13-31 region are arranged in such a way that no definite continuous hydrophobic zone could be formed in an alpha- or pi-helical conformation of this region. In aqueous buffered solutions, peptide 4 showed almost the same amount of alpha-helical structure as beta-endorphin, with a slight tendency toward less helicity in 50% aqueous 2,2,2-trifluoroethanol. In rat brain homogenate, peptide 4 was degraded slightly slower than beta-endorphin, in contrast to the apparently much higher stability of previous models under the same conditions. With regard to opiate receptor binding, peptide 4 was twice as potent as beta-endorphin in mu-receptor assays but half as potent in delta-receptor assays. The opiate potency of peptide 4 on the guinea pig ileum was higher than that of beta-endorphin. In contrast, in the rat vas deferens assay, which is very specific for beta-endorphin, the potency of peptide 4 was very low and could be shown not to be mediated by the same opiate mechanism or by the same opiate receptor. A comparison of these results with those of previous model peptides provides further evidence for the importance of an amphiphilic helical structure in beta-endorphin residues 13-31, which determines the resistance to proteolysis of the natural molecule and contributes to the delta- and mu-opiate receptor interaction. The amphiphilicity of this helical structure must also be essential for high opiate activity on the rat vas deferens (epsilon-receptors), whereas no such structural requirement appears to be necessary for interaction with the opiate receptors on the guinea pig ileum.     

Human beta-endorphin. Immunoreactivity of synthetic analogs with various chain lengths.

Immunoreactivity of synthetic human beta-endorphin analogs with various chain lengths has been examined using a specific radioimmunoassay. It was found that beta-endorphin-(1--21) and analogs of shortened chain exhibit no immunoreactivity, whereas beta-endorphin-(1--15) possesses significant in vitro opiate activity. It appears that immunoreactivity of beta-endorphin resides in the COOH-terminal segment of residues (22--31). The data also show the lack of correlation between opiate and immunological activities of beta-endorphin.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Opioid activity of delta O-endorphin in the guinea pig ileum.

The oligopeptides beta- and delta O-endorphin were isolated from porcine and bovine pituitary respectively. Their opiate activity was determined in the guinea pig ileum and compared to that of the pentapeptide methionine-enkephalin and morphine. The rank order of opioid activity was found to be: morphine greater than beta-endorphin = Met-enkephalin greater than delta O-Endorphin which lacks the four C-terminal amino acids of beta-endorphin displayed 60% of the activity of beta-endorphin. These results indicate, that C-terminal amino acids contribute little to the affinity of beta-endorphin for opiate receptors in the guinea pig ileum.     

Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch.

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti-epilepsy effect in rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP). Its amino-acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3' and 5' RACE methods. It encodes a precursor of 85 amino-acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly-Lys-Lys at the C-terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C-terminus. C-terminal amidation was also verified by the molecular-mass determination of BmK AEP. This anti-epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

Interaction of iodinated human [D-Ala2]beta-endorphin with opitate receptors.

The interaction of beta-endorphin with opiate receptors was studied by using the radioiodinated, metabolically stable D-Ala2 derivative of human beta-endorphin. This analog binds specifically to rat brain membrane preparations with an apparent Kd of about 2.5 x 10-9 M. The ability of various enkephalin analogs, as well as opiate agonists and antagonists, to inhibit the binding of beta-endorphin clearly demonstrates that this peptide can bind to opiate receptors. However, the effects of various cations on the binding of 125I-[D-Ala2]beta-endorphin are markedly different from those found for enkephalin binding. Sodium ion at physiological concentrations decreases substantially the binding of enkephalins but only slightly decreases endorphin binding, whereas manganese enhances enkephalin binding but has no effect on endorphin binding. Moreover, potassium (100 mM) decreases the binding of beta-endorphin but does not affect enkephalin binding. These results suggest that beta-endorphin and enkephalin bind differently to the same receptor or bind to different receptors with overlapping specificity.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Opioid receptor selectivity of peptide models of beta-endorphin

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.     

Functional properties of covalent beta-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation

The 31-residue neuropeptide porcine beta-endorphin was shown to inhibit the Ca2+-dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to beta-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular beta-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. We found that relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking beta-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked beta-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. In contrast, the 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 beta-endorphin 13-31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+-dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.

Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescence changes in the Src homology 2 (SH2) domain. Affinities are in the nanomolar range, the Shc peptide having the highest affinity, followed by peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affinities. Proline-rich peptide binding to the SH3 domains induces fluorescence changes mainly in the C-terminal SH3. Affinities are in the micromolar range, the highest affinity peptides mimicking the first proline-rich motif of the Sos C-terminus. Additional residues before this PVPPPVPP motif provide a minor contribution to the binding, but the two residues after this motif are important and may contribute to specificity. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting that Grb2 recognizes other structural features in the Sos C-terminus. Binding of a phosphotyrosine peptide to the SH2 has no effect on the SH3s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange factor activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.     

Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction: kinetic analysis of inhibition studies

The active complex of Escherichia coli ribonucleotide reductase comprises two dissociable, nonidentical homodimeric proteins, B1 and B2. When B2 is the varied component, the reductase activity is competitively inhibited by synthetic peptides of varying lengths corresponding to the C-terminus of protein B2. This finding provides the first evidence that the C-terminal peptides and protein B2 share the same binding domain on protein B1. Our data also show that two molecules of peptide can bind to protein B1 with equal affinity. Similar inhibition constants (18 microM) were obtained for peptides containing the C-terminal 20, 30, and 37 residues. When the invariant residue Tyr 356 was omitted, a 2-fold decrease in peptide inhibitory ability was observed. A small peptide, lacking the last 11 residues, had virtually no inhibitory potency. These results, coupled with our previous observations that truncated protein B2, in which one or both polypeptide chains are missing approximately 24 C-terminal residues, had considerably lower or no affinity for B1, suggest that the C-terminal regions are the major determinants in the B1-B2 interaction. In the Appendix, two methods for treatment of kinetic situations pertinent to the ribonucleotide reductase system are presented. One method deals with the determination of kinetic parameters for two components present at comparable levels; the other is concerned with the differentiation of linear and nonlinear competitive inhibition involving the binding of two inhibitor molecules. Both methods should find application to other similar cases.     

C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development

Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.     

Structure, dynamics, and selectivity of the sodium channel blocker mu-conotoxin SIIIA

mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.