Deprotonation of a Single Amino Acid Residue Induces Significant Stability in an α-Helical Heteropeptide.

Stability of secondary structural elements is an integral component of a structurally stable protein. Presence of protons in the residue sequence and their immediate environment play a significant role in conformational stability. In this study, we show that removing a proton from a single amino acid residue significantly increases the stability of an α-helical heteropeptide in comparison with the unprotonated form. Far-UV circular dichroism spectroscopy, fluorescence spectroscopy, fluorescence energy transfer measurements, and over 10 μs of all-atom molecular dynamics simulations are used to provide an atomically detailed characterization of this event. There is a single histidine residue in the studied α-helical peptide sequence toward the N-terminal that interacts with a tryptophan located four residues away and quenches the fluorescence when protonated. Removing a proton from this histidine residue dequenches the tryptophan fluorescence and contributes to a significant increase in the helix stability. Atomically detailed analysis of individual residue conformations shows that the protonated histidine tends to be in closer proximity to the tryptophan, which correlates with higher helix content in the N and C termini and lower helix content in the central region of the peptide. In the presence of a neutral histidine, when tryptophan fluorescence is no longer quenched and histidine moves further away from tryptophan, the helix content remains mostly unchanged in the N-and-C termini and significantly increases in the central region. Our results strongly suggest that interactions of the tryptophan with a protonated histidine downregulate the helix population in the central segment of the helical structure compared to a neutral histidine residue. Upregulation of helix population of the central segment of this α-helical heteropeptide in the presence of a neutral histidine residue significantly increases the peptide structural stability.