Fluctuating β‐Sheet Secondary Structure in DS119 Explains the Small Effects of Backbone N‐Amination on Thermal Stability

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.