Redox active tyrosine residues in biomimetic beta hairpins

Biomimetic peptides are autonomously folding secondary structural units designed to serve as models for examining processes that occur in proteins. Although de novo biomimetic peptides are not simply abbreviated versions of proteins already found in nature, designing biomimetic peptides does require an understanding of how native proteins are formed and stabilized. The discovery of autonomously folding fragments of ribonuclease A and tendamistat pioneered the use of biomimetic peptides for determining how the polypeptide sequence stabilizes formation of alpha helices and beta hairpins in aqueous and organic solutions. A set of rules for constructing stable alpha helices have now been established. There is no exact set of rules for designing beta hairpins; however, some factors that must be considered are the identity of the residues in the turn and non-covalent interactions between amino acid side chains. For example, glycine, proline, aspargine, and aspartic acid are favored in turns. Non-covalent interactions that stabilize hairpin formation include salt bridges, pi-stacked aromatic interactions, cation-pi interactions, and hydrophobic interactions. The optimal strand length for beta hairpins depends on the numbers of stabilizing non-covalent interactions and high hairpin propensity amino acids in the specific peptide being designed. Until now, de novo hairpins have not previously been used to examine biological processes aside from protein folding. This thesis uses de novo designed biomimetic peptides as tractable models to examine how non-covalent interactions control the redox properties of tyrosine in enzymes.;Photosystem II provides an example of how non-covalent interactions may alter the redox properties of tyrosine. Photosystem II contains two redox active tyrosine residues, Tyr 161 in the D1 polypeptide and Tyr 160 in the D2 polypeptide. Both Tyr 161 and Tyr 160 contain hydrogen bonds to nearby histidines, yet the two tyrosines are functionally distinct. Tyr 161 mediates electron transfer between the primary donor, P680+. Tyr 160 is involved in assembly of the manganese cluster. Moreover, Tyr 160 and Tyr 161 have different midpoint potentials and their tyrosyl radicals have different lifetimes. The 2.9 A crystal structure of photosystem II reveals that a pi-cation interaction between Tyr 160 and neighboring arginine residue (Arg 272CP47) may contribute to the difference in redox properties of Tyr 160 and Tyr 161.;We have incorporated tyrosine into five de novo designed biomimetic beta hairpin peptides: peptide A, peptide C, peptide D, peptide E, and peptide F. The amino acid content of the peptides was systematically altered to determine how non-covalent interactions with amino acids modulate the redox properties of tyrosine in enzymes. In peptide A, tyrosine is involved in an aromatic interaction with histidine, a hydrogen bond with arginine, and a pi-cation interaction with a second arginine. These non-covalent interactions were varied by single or double amino acid substitution in peptides C through F, and the concomitant alterations in the redox properties of tyrosine were analyzed by EPR spectroscopy, electrochemical titration, and optical titration.;Electrochemical titration of peptide A revealed that oxidation of tyrosine is coupled with protonation of histidine. Substitution of histidine by cyclohexylalanine (peptide C) or by valine (peptide D) eliminated this PCET reaction. Electrochemical titration of peptide A also showed that the aromatic interaction between tyrosine and histidine is associated with a 50 mV decrease in the redox potential of tyrosine. However, removal of the hydrogen bond with arginine (peptide F) or the pi-cation interaction (peptide E) reversed this decrease in redox potential.;Optical titrations were used to predict the pK of tyrosine in peptides A through F. The pKs of tyrosine in peptides A, C, D, and F were indistinguishable. Removal of the Tyr 5-Arg 16 hydrogen bond (Peptide E) decreased the pK of tyrosine from 9.3 to 8.3 and caused small changes in the EPR spectrum of peptide E at pH 5.0.;These data demonstrate that the proton transfer to histidine, the hydrogen bond to arginine, and the pi-cation interaction create a peptide environment that lowers the midpoint potential of tyrosine. Moreover, these interactions contribute equally to control the midpoint potential. The data also show that hydrogen bonding is not the sole determinant of the midpoint potential of tyrosine. Finally, the data suggest that the Tyr 160D2-Arg 272CP47 pi-cation interaction contributes to the differences in redox properties between Tyr 160 and Tyr 161 of photosystem II.