Photochemical Protein Scissors: Role of Aromatic Residues on the Binding Affinity and Photocleavage Efficiency of Pyrenyl Peptides

Abstract

Photocleavage of proteins by a series of organic probe molecules is examined as a function of probe structure. For example, pyrenyl peptides Py-Gly-X (X=Trp, Tyr, Phe, and His, and Py=4(1-pyrenyl)butyroyl)) are prepared, and their protein binding/photocleavage properties have been examined. The binding constants with bovine serum albumin (BSA) are in the range of 107 to 105 and binding of the probes to the proteins is evident in absorption, fluorescence and circular dichroism experiments. While the fluorescence of Py-Gly-Tyr increases upon binding to BSA, quenching is observed with Py-Gly-Trp or Py-Gly-His. While hyperchromism is observed with Py-Gly-Trp/BSA, hypochromism is the norm for all the other probes with this protein. Binding of all the probes to BSA or lysozyme resulted in major changes in the circular dichroism spectra of the probes. Photoexcitation of the probe/protein complexes, in the presence of an electron acceptor, resulted in protein photocleavage. The phenylalanine and histidine analogs resulted in photocleavage of both BSA and lysozyme while the tyrosine and tryptophan analogs did not yield any fragmentation with either of the two proteins. The photocleavage sites are similar to those reported for Py-Phe. Flash photolysis studies of the probe/protein mixtures indicate that the initially produced pyrene cation radical is strongly quenched by the tyrosine and tryptophan residues and resulted in the corresponding amino acid radicals. Strong modulation of the photoreactivities of the probes by specific residues of the probe provide insight in learning how the photocleavage efficiencies of the probes can be improved in future studies.