Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two‐dimensional NMR and CD spectroscopies and constrained molecular dynamics

Abstract

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.

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH2: Systematic Structure−Activity Relationship Studies

“…The observation that 11 Ala-substituted analogues (mostly replacing hydrophilic residues) exhibited potency equal to or greater than that of the parent compound suggests that overall R-helicity and/or hydrophobicity are important factors in receptor binding. Other studies mentioned earlier [61][62][63] showed optimal lactam sizes of 20-and 21-membered rings for retention or gain in biological potencies. In contrast, we saw maximal potencies for a range of ring sizes from 18-to 20-membered [i-(i+4)] and 18-membered [i-(i+3)] lactam analogues in the C-terminus of GHRH(1-29)-NH 2 .…”

Conformation of the Ras-binding domain of Raf studied by molecular dynamics and free energy simulations

“…This is the same model used in the NMR structure determination. 35 All other simulations included explicit solvent, described by a modified TIP3P water model; 36,37 the water internal geometry was constrained with the SHAKE algorithm. 38 Simulations were done with two different force fields for the protein: the CHARMM19 39 and the OPLS/AMBER 40 force fields.…”

Contribution of Conformationally Constrained Calcitonin (Ct) Analogs to the Understanding of the Structural and Conformational Requirements of Calcitonin Bioactivity and to the Design of Potent Agonists

“…In all three peptides, the bridge consisted of an Asp i , Lys i+4 pair that generated a 20-membered ring. Such ring structures had been expected, from previous studies with peptide hormones and model peptides, to stabilize an α-helix [92][93][94][96][97][98][99][100]. To compare the effect of the location of the lactam bridge in a potential α-helical region for structure and bioactivity, analog A1 contained the ring structure at the C-terminal half and analog A3 at the N-terminal half of the potential α-helical region.…”

“…In the past 25 years, the introduction of (i, i+4) side chain-to-side chain lactam bridges into potential α-helical regions has been an approach that has been successfully applied for the stabilization of the α-helical conformation in conformationally flexible polypeptide hormones including growth hormone releasing factor (GRF), neuropeptide Y (NPY), and parathyroid hormone (PTH) [1,6,[91][92][93][94][95][96][97]. According to this approach, specific residues at positions i and i+4 in the putative α-helical region are exchanged for specific diamino-and/or dicarboxylic amino acid residues, such as, for example, Lys or Orn and Asp or Glu, depending on the size of the ring structure to be generated, and the side chains of these residues are connected into a lactam bridge [91].…”