Diproline Templates as Folding Nuclei in Designed Peptides. Conformational Analysis of Synthetic Peptide Helices Containing Amino Terminal Pro-Pro Segments RaiRajkishor AravindaSubrayashastry KanagarajaduraiKaruppiah RaghothamaSrinivasarao ShamalaNarayanaswamy BalaramPadmanabhan 2006 The effect of N-terminal diproline segments in nucleating helical folding in designed peptides has been studied in two model sequences Piv-Pro-Pro-Aib-Leu-Aib-Phe-OMe (<b>1</b>) and Boc-Aib-Pro-Pro-Aib-Val-Ala-Phe-OMe (<b>2</b>). The structure of <b>1</b> in crystals, determined by X-ray diffraction, reveals a helical (α<sub>R</sub>) conformation for the segment residues 2 to 5, stabilized by one 4→1 hydrogen bond and two 5→1 interactions. The N-terminus residue, Pro(1) adopts a polyproline II (<i>P</i><sub>II</sub>) conformation. NMR studies in three different solvent systems support a conformation similar to that observed in crystals. In the apolar solvent CDCl<sub>3</sub>, NOE data favor the population of both completely helical and partially unfolded structures. In the former, the Pro-Pro segment adopts an α<sub>R</sub>-α<sub>R</sub> conformation, whereas in the latter, a <i>P</i><sub>II</sub>-α<sub>R</sub> structure is established. The conformational equilibrium shifts in favor of the <i>P</i><sub>II</sub>-α<sub>R</sub> structure in solvents like methanol and DMSO. A significant population of the Pro(1)-Pro(2) <i>cis</i> conformer is also observed. The NMR results are consistent with the population of at least three conformational states about Pro-Pro segment:  <i>trans</i> α<sub>R</sub>-α<sub>R</sub>, <i>trans</i> <i>P</i><sub>II</sub>-α<sub>R</sub> and <i>cis</i> <i>P</i><sub>II</sub>-α<sub>R</sub>. Of these, the two <i>trans</i> conformers are in rapid dynamic exchange on the NMR time scale, whereas the interconversion between <i>cis</i> and <i>trans</i> form is slow. Similar results are obtained with peptide <b>2</b>. Analysis of 462 diproline segments in protein crystal structures reveals 25 examples of the α<sub>R</sub>-α<sub>R</sub> conformation followed by a helix. Modeling and energy minimization studies suggest that both <i>P</i><sub>II</sub>-α<sub>R</sub> and α<sub>R</sub>-α<sub>R</sub> conformations have very similar energies in the model hexapeptide <b>1</b>.