Lipid-induced conformational changes in glucagon,secretin, and vasoactive intestinal peptide

The CD of glucagon, secretin, and vasoactive intestinal peptide has been studied as a function of temperature in water and in aqueous solutions of dodecyl sulfate, phosphatidyl glycerol, and L -α-phosphatidic acid (dipalmitoyl). The anionic detergent and lipids induce helix formation in all three peptides, with the amount of induced helical content increasing in the order glucagon < secretin < vasoactive intestinal peptide. These observations are subject to quantitative rationalization using a matrix formulation for the configuration partition function. In this formulation the major conformational consequences of the interaction with anionic lipids or detergents is an increase in the probability for helix formation by arginyl, histidyl, and lysyl residues. The region in which helix formation is maximal is found to be at amino acid residues 13–20 in all three peptides. Other studies have implicated this portion of the polypeptide chain in receptor binding. Thus, the helical segment induced by interaction with anionic lipids may play an important physiological role.     

Helicity of short E-R/K peptides

Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide‐based drug design. Previous studies of synthetic Ala‐based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i + 3) or four (i, i + 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E‐R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E‐R/K–based peptides show significant helix content. Peptides containing one or more E‐R interactions display greater helicity than those with similar E‐K interactions. Significant helicity is achieved in Arg‐based E‐R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i + 3 and i + 4 salt bridge has substantial contribution to fractional helix content. The E‐R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.     

Peptidic HIV integrase inhibitors derived from HIV gene products: Structure–activity relationship studies

Structure–activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an α-helical conformation, Glu-Lys pairs were introduced into the i and i + 4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an α-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.     

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb–Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb–Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ ₁ dihedral combinations (g+, g+) and (g?, g+), respectively. These low energy conformations were also observed in intrahelical Asp–Arg and Glu–Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.     

Identification of secretin,vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.     

Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum

Summary The digestive tract of the cephalochordate Branchiostoma lanceolatum was investigated with regard to occurrence and distribution of endocrine cells. By the use of the peroxidase-antiperoxidase (PAP) technique, cells in the gut epithelium reacting with antisera against 8 different mammalian polypeptide hormones were localized. Positive reactions were obtained with antisera against the four mammalian islet hormones (insulin, glucagon, pancreatic polypeptide, somatostatin) and against secretin, vasoactive intestinal polypeptide, pentagastrin and neurotensin. No immunoreactivity was found with antisera against members of the lipotropin family (ACTH, met-enkephalin, -endorphin), against big-gastrin, cholecystokinin, substance P and moulin. The exact mapping of the different polypeptide immunoreactive cells throughout the digestive tract of Branchiostoma lanceolatum is presented.     

Stimulation of adenylate cyclase from isolated hepatocytes and Kupffer cells.

Hepatocytes and Kupffer cells were separated from rat liver after prelabeling the Kupffer cells with colloidal iron and perfusion of the liver with digestive enzymes. The activity of several enzymes from Kupffer cells and hepatocytes was compared to validate this method of cell separation. The ratios of hepatocyte to Kupffer cell specific activities of glucose-6-phosphatase, 5'-nucleotidase, adenylate cyclase, and acid phosphatase were 20, 0.39, 0.18, and 0.078, respectively. Adenylate cyclases from hepatocytes and Kupffer cells were stimulated by fluoride ion, GTP, and catecholamines. Hepatocyte adenylate cyclase was also stimulated by glucagon, secretin, vasoactive intestinal polypeptide, and by prostaglandin E1, whereas, the Kupffer cell enzyme was completely insensitive to these hormones. The stimulation of hepatocyte adenylate cyclase by combinations of glucagon plus secretin, or glucagon plus vasoactive intestinal polypeptide, were equivalent to the sum of the individual stimulations. This suggests that the hepatocyte has specific receptors for glucagon and for vasoactive intestinal polypeptide and secretin. Prostaglandin E1 stimulation of hepatocyte adenylate cyclase was not additive to the stimulation caused by polypeptide hormones or catecholamines, nor did prostaglandin E1 decrease stimulation caused by these hormones. Although prostaglandin-sensitive adenylate cyclase was recovered with hepatocytes, 40 to 50% of the total liver prostaglandin-sensitive activity was recovered in a fraction of cell debris mixed with small cells which did not phagocytize colloidal iron.     

Transformation of the ϕ-ψ plot for proteins to a new representation with local helicity and peptide torsional angles as variables

A new representation of protein structure is obtained by the angular coordinate transformations η_i = (?_i+1?ψ_i)/2 and ξ_i = ?_i+1+ψ_i with careful mathematical attention to the cyclical boundary conditions of all of the variables involved. From published ?-ψ data it is possible to obtain a new η-ξ plot. As the angle ξ_i is varied from – 180° through 0° to + 180° in this plot, the local helicity of the polypeptide chain changes continuously and contiguously without sudden reversals in handedness. The variable, η_i, gives the torsional position of the ith peptide group. Some peptide groups in proteins, such as the second peptide residue in a type II β-turn, are nonhydrogen-bonded and can undergo considerable torsional oscillation. In such cases the η angle should be represented by a line whose length reflects the allowed dynamical variations in the peptide torsional position. Certain peptide residues in proteins may be able to undergo a complete torsional rotation of 360°. Such residues would be represented on the η-ξ plot as a straight line across the plot parallel to the abscissa. Other examples of the possible usefulness of this plot are also given.     

Binding of hormones and neuropeptides by calmodulin

Calmodulin exhibits high-affinity, calcium-dependent binding of 1 mol/mol of the vasoactive intestinal peptide (VIP), secretin, and either the 42- or 43-residue gastric inhibitory peptide (GIP) with dissociation constants of 0.05-0.14 microM. The affinity of VIP for calmodulin approaches its affinity for the cell-surface VIP receptors. These peptides compete with both smooth muscle myosin light chain kinase and glucagon in calmodulin binding. Calculation of amino acid frequencies for eight calmodulin binding peptides (VIP, GIP, secretin, ACTH, beta-endorphin, substance P, glucagon, and dynorphin [Malencik, D. A., & Anderson, S. R. (1982) Biochemistry 21, 3480]) shows a below-average incidence of glutamyl residues, above-average incidence of glutaminyl residues, and average incidence of both aspartyl and asparaginyl residues. Predictions of structure from sequence suggest that the bound peptides contain strongly basic turns and coils in close association with regions having above-average beta-sheet potential. The temperature dependence of glucagon binding by calmodulin shows that the association is enthalpy driven.     

Regulatory peptides in the gastrointestinal tract of Alligator mississipiensis

Summary The gastrointestinal tract of the alligator Alligator mississipiensis has been investigated for the presence of immunoreactivity to fourteen regulatory peptides all known to occur in the mammalian gut system.Mucosal endocrine cells reacting specifically with the antisera to neurotensin, C-terminal gastrin, somatostatin, bombesin, secretin, pancreatic glucagon and enteroglucagon were detectable, the distribution of these cells being, in general, similar to the mammalian pattern. Peripheral nerve cell bodies and nerve fibres were detected with the antisera to vasoactive intestinal polypeptide, substance P, bombesin and somatostatin again with a distribution similar to that seen in mammals.No immunoreactivity was observed with the available antisera to glicentin, motilin, gastric inhibitory polypeptide, gastrin 34, cholecystokinin 9–20 and met-enkephalin.     

Accommodation of a D-Phe residue into a right-handed 310-helix: Structure of Boc-D-Phe-(Aib)4-Gly-L-Leu-(Aib)2-Ome,an analogue of the amino terminal segment of antiamoebins and emerimicins

The crystal structure of the nonapeptide Boc-D -Phe-Aib-Aib-Aib-Aib-Gly-Leu-Aib-AibOMe (I), which is an analogue of the N-terminal sequence of antiamoebins and emerimicins, establishes a completely 3₁₀-helical conformation with seven successive intramolecular 4 → 1 hydrogen bonds. The average, ?, ψ values for residues 1–8 are ?59° and ?32°, respectively. Crystal parameters are C₄₇H₇₇N₉O₁₂, space group P1, a = 10.636(4) Å, b = 11.239(4) Å, c = 12.227(6) Å, α = 101.17(4)°, β = 97.22(4)°, γ = 89.80(3)°, Z = 1, R = 5.95% for 3018 data with |F₀| > 3α(F), resolution 0.93 Å. The use of the torsion angle κ = C(i ? 1)N(i)C^α(i)C^β(i), where κ = 68° for D -Phe and κ = 164° for L -Leu, confirms the opposite configurations of these residues. The ?, ψ values of ?62° and ?32° at D -Phe are unusual, since this region is characteristic of residues with L configurations. Peptide I possesses only two chiral residues of opposing configuration. The observed right-handed 3₁₀-helical structure suggests that helix sense has probably been determined by the stereo-chemical preferences of the Leu residue. © 1993 John Wiley & Sons, Inc.     

Effects of secretin and gastric inhibitory polypeptide on human pancreatic growth hormone-releasing factor(1-40)-stimulated growth hormone levels in the rat

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.     

Interactions of human secretin with sterically stabilized phospholipid micelles amplify peptide-induced vasodilation in vivo

Secretin, a 27-amino acid neuropeptide, is a member of the glucagon/secretin/vasoactive intestinal polypeptide (VIP) superfamily of amphipathic peptides that elicits transient vasodilation in vivo. The purpose of this study was to determine whether association of human secretin with sterically stabilized phospholipid micelles (SSM) amplifies the vasorelaxant effects of the peptide in the peripheral microcirculation in vivo. We found that secretin in saline evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch microcirculation (P < 0.05). This response was potentiated and prolonged significantly when secretin was associated with SSM (P < 0.05). Vasodilation evoked by secretin in saline and secretin in SSM was abrogated by VIP(10-28), a VIP receptor antagonist, but not by PACAP(6-38), a PACAP receptor antagonist, or Hoe140, a selective bradykinin B(2) receptor antagonist. Collectively, these data indicate that self-association of human secretin with SSM significantly amplifies peptide vasoreactivity in the intact peripheral microcirculation through activation of VIP receptors. We suggest that the vasoactive effects of human secretin in vivo are, in part, phospholipid-dependent.     

Resonant recognition model of neuropeptide Y family: hot spot amino acid distribution in the sequences

The resonant recognition model is used to predict structurally and functionally important amino acid residues (so-called hot spots) in the neuropeptide Y (NPY) family. Thirty-three polypeptides belong to this family. All of them consist of 36 amino acids. The model predicts that residues 10 and 28 in the polypeptides are hot spots. In the 33 polypeptides, most of the amino acids at residue 10 are acidic amino acids, glutamic acid and aspartic acid. Other minor amino acids, serine, glycine, and proline, have high probabilities of -turn occurrence. Amino acids at residue 28 are all branched hydrophobic amino acids, isoleucine, leucine, and valine. The profile for predicting hot spots indicates repeating patterns of residues 1–18 and residues 19–36. Absolute values at residue i and residue i + 18 are the same, but these residues have opposite signs. Therefore the model of the NPY family predicts hot spots concerning a combination of residue i and residue i + 18.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

Cloning and Sequence Analysis of cDNA for Diuretic Hormone Receptor from the Bombyx mori

The insect diuretic hormone (DH) binds to their receptor in malpighian tubules, and stimulates water secretion and cAMP synthesis. Complementary DNA encoding a diuretic hormone receptor was cloned from the malpighian tubules of Bombyx mori. The cloned cDNA encodes a protein consisting of 391 amino acid residues with the seven transmembrane domains. The receptor protein is homologous with that of other insects, and is structurally related to G-protein coupled receptors such as corticotropin relating factor (CRF), secretin, and vasoactive intestinal peptide.     

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective ‘unlabeling’ or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly ¹³C/¹⁵N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {¹²CO _i –¹⁵N _i+1}-filtered HSQC, which aids in linking the ¹H^N/¹⁵N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i − 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to ²H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of ¹⁴N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.     

The endocrine pancreas of Alligator mississippiensis

Summary Immunocytochemical methods for light and electron microscopy were used to demonstrate the regulatory peptides present in the endocrine pancreas of thealligator, Alligator mississippiensis.The peptides studied included insulin, glucagon (pancreatic and enteric), somatostatin, pancreatic polypeptide (avian, bovine and human), vasoactive intestinal polypeptide, substance P, metenkephalin, -endorphin, C-terminal gastrin/CCK and gastric inhibitory polypeptide. Endocrine cells were detected using antisera to insulin, pancreatic glucagon, somatostatin and avian pancreatic polypeptide, whereas peptidergic nerves were stained with antisera to vasoactive intestinal polypeptide. All other antisera were unreactive in the alligator pancreas. The peptide-containing structures were identified ultrastructurally by both the semithin/thin and immuno-gold methods. The results showed that five of the regulatory peptides commonly detected in the mammalian pancreas were immunologically recognisable in the alligator. In addition, the ultrastructural appearance of the peptide-containing cells was clearly distinct from that reported in mammals.     

Triangular matrix representation of dimensionless helical hydrophobic moment ratios

If the hydrophobicity of the ith residue in an α-helix is denoted by H_i, one can (following Eisenberg) define a corresponding vector, h_i = a_iH_i. Here a_i is a unit vector directed radially from the helix axis through C_i^α. The vector sum of the h_i for the N residues in an α-helix is denoted by h, and h² is the square of the magnitude of h. In the absence of any correlation in the h_i we obtain h² = ∑ H_i², where the sum extends over all N residues in the helix. The dimensionless ratio h²/∑ H_i² has the value 1 when the h_i are uncorrected. However, if hydrophobic residues occur on the opposite surface of a helix from the hydrophilic residues, h²/∑ H_i² > 1. The h²/∑ H_i² for the n(n − 1)/2 helices that might occur in a chain of n residues are conveniently displayed as the elements in a symmetric n × n matrix. The jkth and kjth elements are h²/∑ H_i² for the helix that initiates at residue j and terminates at residue k. All of the h²/∑ H_i² are retained in either of the triangular matrices that together together constitute the symmetric matrix. The largest element in the triangular matrix for glucagon is the entry for a helix that is nearly identical with the major helix found by Wüthrich when the peptide is bound to a zwitterionic micelle. Three related hormonal peptides have their maximum h²/∑ H_i² in the same region of their respectively triangular matrices. The triangular matrix for α-tropomyosin shows that the h²/∑ H_i² are larger in the portion of the chain that forms the more stable helix in the in-register dimer.