A novel cyclic GRF analog, cyclo(Asp8‐Lys12)‐[Asp8,Ala15]‐GRF(1‐29)‐NH2, i.e. cyclo8.12[Asp8,Ala15]‐GRF(1‐29)‐NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (∼2h) using the BOP reagent. Substitution of Ala12 with d‐Ala2 and/or NH2‐terminal replacement (desNH2‐Tyr1 or N‐MeTyr1) in the cyclo8.12[Asp8,Ala15]‐GRF(1‐29)‐NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE‐derived distance constraints) demonstrated that cyclo8.12[Asp8,Ala15]‐GRF(1‐29)‐NH2 contains a long α‐helical segment even in aqueous solution. A series of cyclo8.12 stereoisomers containing d‐Asp8 and/or d‐Lys12 were prepared and also found to be highly potent and to retain significant α‐helical conformation. The high biological activity of cyclo8.12[N‐MeTyr1,d‐Ala2,Asp8,Ala15]‐GRF(1‐29)‐NH2 may be explained on the basis of retention of a preferred bioactive conformation.
Structures have been determined for a potent analogue of vasoactive intestinal peptide (VIP), Ac-[Lys12, Lys14, Nle17, Val26, Thr28]VIP (VIP'), in methanol/water solutions. In CD studies, both VIP and VIP' were helical in methanol/water, with the percentage of alpha-helix increasing with percentage methanol. The pH had little effect on the structure. Complete 1H NMR assignments were made for VIP' in 25% methanol at pH 4 and 6 and in 50% methanol at pH 6, using two-dimensional COSY, NOESY, and relay-COSY experiments. There were no widespread changes in chemical shifts between the samples at pH 4 and 6; however, widespread changes were observed between the samples in 25% and 50% methanol. Complete sets of NOEs were obtained for VIP' in 25% methanol, pH 4, and in 50% methanol, pH 6. These NOEs were converted into distance constraints and applied in molecular dynamics and energy minimization calculations using the program CHARMM. A set of low-energy structures was obtained for VIP' in each solvent system. In 25% methanol, VIP' has two helical segments at residues 9-17 and 23-28. The remainder of the structure is not well determined. In 50% methanol, residues 8-26 form a regular, well-defined alpha-helix and residues 5-8 form a type III beta-turn. The remaining residues are not ordered. These structural assessments agree with the CD data. In the lowest energy structure in 50% methanol, the side chains of Asp3, Phe6, Thr7, and Tyr10 are clustered together--these residues are conserved throughout the family of peptide hormones homologous to VIP.
Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70).Substitution of Asp2o or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp2o was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.of the p55/p70 complex (i.e., the high-affinity IL-2R) that appears to be essential for a full proliferative response by T cells and for the late phase of effector lymphokine-activated killer functions.To understand the structure-function relationship ofhuIL-2 and the interactions with its receptor, we have endeavored to identify those amino acids that mediate binding to the high-affinity receptor as well as to its two component subunits. By using site-directed mutagenesis, we had engineered (11) a series of huIL-2 analogs by the introduction of specific deletions and substitutions to modify the protein. The mutant huIL-2 proteins were produced in Escherichia coli and assayed for biological activity. We have now extended these studies with additional analogs and assayed their ability to compete for binding to the IL-2R and its subunits. The identification of specific amino acids required for bioactivity and binding to the p55 and p70 subunits has allowed us to correlate the structure of IL-2 with its biological functions.Human interleukin 2 (huIL-2) exerts immunoregulatory effects on a variety of cells including T cells, activated B cells, natural killer cells, and lymphokine-activated killer cells. The biological effects of huIL-2 are mediated through specific interactions with cell surface receptors present on the target cells (1, 2). High-, intermediate-, and low-affinity forms ofthe IL-2 receptor (IL-2R) have been identified on these cells. The high-affinity receptor exhibits an apparent Kd of =10-1 M and exists as a complex of at least two distinct polypeptides, with molecular masses of 55 kDa (p55 or Tac) and 70-75 kDa (p70 or p75). Cells or cell lines expressing only p55 are capable of binding 4). These cells have been used to demonstrate that the p55 subunit corresponds to the lowaffinity form of the receptor (Kd = 1-3 x 10-8 M). It has been reported that the p70 protein, which is expressed at relatively high levels on the human YT-...
Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.
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