PEPTIDE RULER BASED ON PORPHYRIN TRIPLET STATE FOR DISTANCE DETERMINATION BY PELDOR SPECTROSCOPY

An innovative spin labeling method for distance determination in the range between 15 and 80 Å by Pulse ELectron Double Resonance (PELDOR), exploiting the porphyrin triplet state as paramagnetic probe, has been proposed [1]. This advance Electron Paramagnetic Resonance (EPR) technique was tested on a series of eight α-helical peptides of increasing length. The system was designed as a molecular ruler containing two paramagnetic labels at different distances: the 5-(4- carboxyphenyl)-10,15,20-triphenylporphyrin (TPP) was attached at the N-terminal end of the peptide while the artificial amino acid 4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine- 4-carboxylic acid (TOAC) was inserted at variable positions along the helix. The peptides, composed by alternating L-alanine (Ala) and α-aminoisobutyric acid (Aib) residues, were synthesized by standard solid phase synthesis inserting the TOAC amino acid at the proper position of the chain and covalently linking the TPP on the N-terminal amino group. During the PELDOR experiment the TPP moiety is photoexcited by a laser flash to generate the triplet state which couples to the nitroxide residue via dipolar interaction allowing the distance determination between the two probes. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels.