Coiled coil structure of the cJun-cFos interaction.

<p>Shown are the coiled coil regions of cJun (red) and cFos (blue) interaction (PDB coordinates: 1FOS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059415#pone.0059415-Glover1" target="_blank">[58]</a>). Side-chains for interfacial <b>‘</b><b><i>a’ ‘d’ ‘e’ and ‘g’</i></b> residues are shown and highlight the fact that the interactions between them are distributed broadly across the molecule. Key hydrophobic interfacial side-chains within cJun that are predicted to be required for effective binding to cFos are shown in green (<b><i>d2</i></b>, <b><i>a3</i></b>, <b><i>d3</i></b>, <b><i>a4</i></b>, <b><i>d4</i></b>, <b><i>a5</i></b>, <b><i>d5</i></b>, top to bottom: L, N, L, A, L, V, L).</p

Serum stability of peptides 12 and 24.

<p>Shown are the effects of helix-inducing constraints (• and ▪) versus the linear sequences (▾ and ▴) in human serum at 37°C.</p

Schematic showing sequences and constrains for all peptides.

<p>The parental JunW_CANDI sequence is shown in bold as are heptads and residue positioning within the helical wheels. Peptide constraints are shown in blue. K_D values taken from ITC experiments for peptides in complex with cFos are shown in µM. Fraction helicity as measured from CD experiments are also shown. Positions of <i>i</i>→<i>i+4</i> hydrocarbon constraints were initially placed into a JunW_CANDI peptide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059415#pone.0059415-Mason2" target="_blank">[42]</a> lacking capping motifs, causing a reduction in the size of the molecule from 37 residues to 32. All constraints tethered <i>b</i>→<i>f</i> or <i>f</i>→<i>c</i> residues.</p

Isothermal Titration Calorimetry (ITC) analysis of leucine zipper domain interactions between constrained peptides and cFos.

<p>Shown are isotherms for all ten measureable heterodimers (1, 2, 8, 10, 11, 12, 17, 20, 22, and 24) injected into a cell containinginto cFos. The top and bottom panels show, respectively, raw data after baseline correction. During ITC experiments, approximately 200–600 µM of peptide A was injected in 30–40×5 µl batches from the injection syringe into the cell, which contained 10–40 µM cFos. Both partners were in a 10 mM Potassium Phosphate buffer, 100 mM Potassium Fluoride at pH 7. Experiments were undertaken at 20°C. The solid lines represent the fit of the data to the function based on the binding of a ligand to a macromolecule using the Microcal (GE Healthcare) Origin software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059415#pone.0059415-Wiseman1" target="_blank">[57]</a>.</p

Thermodynamics of binding of cJun analogues to cFos. Columns (from left to right) show i) Tm values from thermal denaturation analysis ii) calculated % helicity for each respective pair calculated from circular dichroism spectra and iii) K_D values calculated from thermal denaturation data.

<p>The remaining three columns give stoichiometry of binding and thermodynamic data calculated from ITC, with TΔS calculated according to the Gibbs Helmholtz equation.</p>*<p>data calculated using the midpoint of the transition from thermal denaturation profiles (and fit as temperature as a function of lnK_D, with the fit lnK_D = aT+C where a is the gradient, T is the temperature in Celsius and C is the intercept) and calculated at 20°C.</p>#<p>Calculated according to TΔS = ΔH−ΔG.</p

Raw thermal melting data for all homo and heterodimeric complexes.

<p>Data have been collected by measuring the level of helicity at 222 nm in an applied photophysics chirascan Circular Dichroism (CD) Spectrometer. Data have been converted from raw ellipticity to Molar Residue Ellipticity (MRE) according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059415#pone.0059415.e001" target="_blank">equation 1</a> to take account of the different peptide lengths. Thermal melting data for cFos is shown in black, data for the constrained peptide in isolation is shown in blue and the cFos/constrained peptide mixture is shown in red. Also shown is the average of the cFos and constrained peptide (black dotted line). Where possible data have been fitted to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059415#pone.0059415.e002" target="_blank">equation 2</a> to generate thermal melting (T_m) values (e.g. for cFos-<b><i>1</i></b> and cFos-<b><i>2</i></b>) and in such instances it is clear from an increase in the averaged homomeric T_m values that an interaction is occurring (e.g. <b><i>1</i></b>: −1+50 = 49/2 = 24.5<55). However, some data were unable to be fitted owing to the lack of a melting transition, or of a lower baseline (e.g. cFos-<b><i>3</i></b> and cFos-<b><i>4</i></b>), indicating that an interaction is not occurring. CD spectra for these pairs are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059415#pone.0059415.s003" target="_blank">Figure S3</a>.</p

NMR Structure of peptide 24.

<p><b>a)</b> NOE summary diagram for peptide <b>24</b> in 90% H₂O:10% D₂O at 298 K. Sequential, short and medium range NOE intensities were classified as strong (upper distance constraint 2.7 Å), medium (3.5 Å), weak (5.0 Å), very weak (6.0 Å) and are proportional to bar thickness; grey bars indicate overlapping signals. ³<i>J</i>_NHCHαcoupling constants <6 Hz are indicated by ↓. Amide NH’s for which chemical shifts changed by <5 ppb/K are indicated by •. <b>b)</b> Backbone superimposition for ten lowest energy NMR-derived solution structures for Ac-cyclo-(3,7; 10,14; 17,21)-ChaR[KEIYD]LR[KKAND]LR[KHIAD]Cha-NH₂ (<b>24</b>) in H₂O:D₂O (9∶1) at 298 K showing carbon atoms (green), nitrogens (blue), oxygens (red), <i>i</i>→<i>i+4</i> hydrocarbon constraints (orange). Also for clarity, one structure is shown with its alpha helical backbone (yellow) and projecting side chains (green). N-terminus is at the top.</p

Un incontro internazionale sugli effetti dell'innalzamento del livello marino

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes

Cyclic Penta- and Hexaleucine Peptides without <i>N</i>‑Methylation Are Orally Absorbed

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with <i>N</i>-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no <i>N</i>-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4–17%) approaching that of the heavily <i>N</i>-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for <i>N</i>-methylation or modified amino acids