Our research group has developed a novel furan-oxidation based crosslinking technology for oligonucleotides,1-6 further applicable to the investigation of peptide-protein interactions.7,8 Furan, a small aromatic compound, is incorporated in peptides using 2-furyl-L-alanine, which can be oxidized to a reactive aldehyde producing singlet oxygen upon irradiation of a photosensitizer such as Rose Bengal and Rhodamine. Furan crosslinking enables formation of a site-selective crosslink between biological binding partners. Upon oxidation of the furan moiety, a crosslinking reaction with sulfhydryl and amine groups present in the protein occur if sufficiently proximate. As the furan moiety is isosteric with histidine and isoelectronic with tyrosine, the incorporation in peptides is well tolerated. Here, we have optimized a fast and efficient formation of a selective crosslink between actin and different furan analogues of the actin binding protein thymosin ß4(Tß4).
Actin is the major cytoskeletal protein of the cell and forms filaments upon polymerization.9 Actin binding proteins (ABPs)10 regulate the polymerization of actin and keep the actin monomeric by interacting with G-actin. Tβ4 is the most abundant of these actin-sequestering proteins, preventing G-actin association to both ends of actin filaments. The furan-modified Tß4 analogues were still able to sequester monomeric actin with comparable or slightly higher capacity than wildtype Tß4. The position of the furan was rationally chosed based on known crystal structures of the actin-Tß4 complex, to ensure the proximity of a nucleophilic residue to facilitate crosslink formation.
Using the furan crosslinking technology, we were able to efficiently crosslink the different furan Tß4 analogues to monomeric actin using mild reaction conditions with low irradiation times and preventing degradation of the binding partners.