Fluctuating β‐sheet secondary structure in DS119 explains the small effects of backbone N‐amination on thermal stability

Journal article

The miniprotein DS119 has been used as a model system to probe the effects of introducing residues with backbone N-amination into a region of parallel β-sheet secondary structure. Derivatives featuring backbone N-methylation have also been synthesized for comparison. As expected, N-methylation of Trp9 or Phe33, positioned on the outer edge of the β-strands, led to a reduction in the thermal stability of the protein. However, contrary to predictions, the N-amination of Trp9 or Phe33 did not lead to an increase in thermal stability of DS119. Refinement of the DS119 structure, using NOE restrained molecular dynamics simulations, shows that the β-sheet region is highly fluctuating in nature. Key interstrand hydrogen bonds have populations of 24%–77%, whereas others have populations of less than 10%. In this disordered β-sheet region, the altered conformational preferences arising from backbone N-amination therefore have only minimal effects. This study demonstrates how MD simulation refinement can identify important dynamical features in a protein structure that might be overlooked in standard protein structure determination protocols.

Authors: Qiao, Y; Starnes, SK; Dolenc, J; Del Valle, JR; Smith, LJ

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Wiley
• Journal: Journal of Peptide Science
• Volume: 32
• Issue: 5
• Article number: e70093
• Publication date: 2026-03-19
• Acceptance date: 2026-03-05
• DOI: 10.1002/psc.70093
• EISSN: 1099-1387
• ISSN: 1075-2617
• Language: English
• Keywords: protein design; molecular dynamics simulation; modified amino acids; Namination; protein dynamics; protein folding