Attenuation of molecular function by multiphoton excitation-evoked chromophore-assisted laser inactivation (MP-CALI) using green fluorescent protein

Abstract

ABSTRACT Noninvasive and straightforward methods to inactivate selected proteins in the living cell with high spatiotemporal resolution are eagerly sought for elucidation of protein func tion in the post-genome-mapping era. Chromophore-assisted laser inactivation (CALI) facilitates inactivation of proteins by photochemically generated reactive oxygen species (ROS), but CALI using single-photon excitation thus far has presented several drawbacks, including its complex procedure, low efficiencies of inactiva tion with a certain chromophore, and phot odamage effects. We here show that by application of multiphoton excitation to CALI using near-inf rared femtosecond laser, enhanced green fluorescent protein (EGFP) can work as an effective chromophore for inactivation of a protein’s function without nonspecific photodamage in the living cell. Keywords: Multi-photon laser scanning microscopy, chromophor e-assisted laser inactivation, green fluorescent protein. INTRODUCTION Enormous amounts of information have been provided by genetic approaches such as gene knockouts and siRNA, which ultimately modify protein expression in the entire cell. However, it is known that the same protein often performs different functions depending on its location. Moreover, a protein serves distinct functions at a particular stage of temporal events. These spatiotemporal di fferences in protein functio ns are difficult to analyze with conventional genetic approaches. The chromophore-assisted lase r inactivation (CALI) method enables us to analyze spatiotemporal functions of the protein in the living cell. Chromophore-assisted laser inactivation Originally the CALI method involves a specific but inert antibody, which is labeled with a chromophore, malachite green, bound to a target protein. Subsequently, laser irradiation generates reactive oxygen species (ROS) of short half-life and thus can specifically inactivate functions of the target protein