Abstract
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.