Investigating the Mechanism of cis Amide Bond Stabilization in Phosphoserine‐Proline Sequences

Phosphorylation is a reversible covalent modification to biomolecules that is central to protein‐protein interactions, signal transduction, and gene expression. When improperly regulated, phosphorylation can trigger oncogenesis and neurodegenerative diseases such as Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Phosphorylation can also act as a structural switch for cis‐trans isomerization of peptide bonds: through intraresidue phosphate‐amide hydrogen bonds, phosphorylated residues promote compact trans conformations. In other contexts, phosphorylated Serine (pSer) residues can promote cis peptide bonds through unknown mechanisms. To investigate the basis for these structural effects, a series of X‐pSer‐Pro‐Z peptides was synthesized with modifications on the proline, N‐terminal to pSer (X), and C‐terminal to Pro (Z). Dipeptides remove influences from secondary and tertiary protein structures, allowing elucidation of key molecular interactions that affect the cis conformer. Two 4 S ‐substituted hydroxyproline derivatives, Fmoc‐ 4 S ‐iodophenyl‐hydroxyproline and Fmoc‐4 S ‐fluoroproline, were synthesized and incorporated into the peptide. These substitutions promote the cis‐ Pro conformation via intraresidue stereoelectronic effects. The initial peptides were examined with C‐terminal amides, while modifications of the N‐terminus allowed for control of sterics. Formyl, acetyl, pivaloyl, 2‐iodobenzoyl, and 4‐iodobenzoyl moieties were included in peptides to provide varying degrees of steric demand to promote alternate conformations at the N‐terminus. The peptides were analyzed by 1 H NMR to quantify the effects of proline, N‐terminal modifications, and phosphorylation on structure. TOCSY and ROESY 1 H NMR spectra were employed to differentiate the cis and trans peaks and characterize the conformational changes. Structural changes as a function of phosphate protonation state were identified by pH‐dependent NMR experiments. The downfield shift in the amide region with increasing pH implied a Hydrogen bonding interaction between the phosphate oxygen and its own amide hydrogen. The equilibrium constant for cis‐trans isomerization, K trans/cis , increased with increasing pH for the acetyl and 4‐iodobenzoyl derivatives. Pivaloyl and 2‐iodobenzoyl derivatives showed an opposite trend, increasing the population of cis with increasing pH. N‐terminal 2‐iodobenzoyl and acetyl groups show greater preference towards the cis at various pH with respect to other examined peptides. Stabilization of cis amide bonds upon phosphorylation can be used to design tunable scaffolds for peptide conformations. Support or Funding Information David M. Heitzer Award, DOD This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .