Year: 2020
Type: article
Abstract: Chromophore Assisted Laser Inactivation (CALI) is a technique that uses irradiation of chromophores proximate to a target protein to inactivate function. Previously, EGFP mediated CALI has been used to inactivate EGFP-fusion proteins in a spatio-temporally defined manner within cells but the mechanism of inactivation is unknown 1, 2. To help elucidate the mechanism of protein inactivation mediated by fluorescent protein CALI ([FP]-CALI), the activities of purified Glutathione-S-transferase-FP (GST-EXFP) fusions were measured after laser irradiation in vitro. Singlet oxygen and free radical quenchers as well as the removal of oxygen inhibited CALI, indicating the involvement of a reactive oxygen species (ROS). At higher concentrations of protein, turbidity after CALI increased significantly indicating cross-linking of proximate fusion proteins suggesting that damage of residues on the surface the protein, distant from the active site, results in inactivation. Control experiments removed sample heating as a possible cause of these effects. Different FP mutants fused to GST vary in their CALI efficiency in the order EGFP>EYFP>ECFP, while a GST construct that binds FlAsH results in significantly higher CALI efficiency than any of the XFPs tested. It is likely that the hierarchy of XFP effectiveness reflects the balance between ROS that are trapped within the XFP structure and cause fluorophore and chromophore bleaching and those that escape to effect CALI of proximate proteins.
DOI: 10.17615/z6zy-5681
Authors: Sligar, Stephen G., Humphrey, David, Yang, Bing, Jacobson, Ken, Rajfur, Zenon, Chen, Zaozao, McLean, Mark A.
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funder: National Institutes of Health
Award: G9000006921
OpenAlex ID: W4297960955