Elucidation of Peptide Bond Diversity using High-resolution Neutron Crystallography

The planarity of the peptide bond plays a crucial role in protein stability and structural formation. However, recent high-resolution structural studies have revealed considerable deviations from ideal peptide bond planarity. In this study, we present a high-resolution neutron structure（1.2 Å resolution）of the oxidized form of high-potential iron-sulfur protein（HiPIP）to investigate peptide bond geometry, including amide protons, with explicit visualization of hydrogen atoms. Our neutron structure clearly showed deviations in the nuclear positions of amide protons from the peptide plane, particularly shifting toward hydrogen bond acceptors. This proton displacement strongly correlates with pyramidalization of the nitrogen atom, which significantly influences the planarity of the H–N–C=O plane. Moreover, the orientation of the amide proton in Cys75 is different between the reduced and oxidized states because of the electron storage capacity of the iron-sulfur cluster. These results show the importance of local conformation in regulating peptide bond geometry. This study demonstrates the unique advantages of high-resolution neutron crystallography in elucidating fine structural details and understanding protein function.