Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.

Abstract

Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALl indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no signicant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.Chromophore-assisted laser inactivation (CALI) of protein function (1) targets laser energy to damage single protein species by binding the protein with a specific antibody that has been labeled with the chromophore malachite green. The bound dye is excited by 620-nm light, a wavelength not significantly absorbed by most cellular components, and damage is localized to within 60 A of the chromophore (2).CALI has been used to show the in vivo roles of several proteins in insect neural development (3-5).Further characterization and understanding of CALI are required for its optimization and judicious application in biological research. A dependence of CALI on the distance from the dye, the laser power, the number of pulses, and the concentration of dye-labeled ligand has been shown (1, 2). Although thermal denaturation was postulated as the molecular mechanism of CALI, the precise nature of the lasermediated damage to the protein function has not been established. This study was undertaken to ascertain the mechanism of protein damage by CALI.The likely sources of photoinduced damage through the absorption of light energy by a chromophore at the radiant flux used for CALI are photothermal and photochemical (6). Photothermal damage would occur through highly localized denaturation of the antibody-antigen complex caused by the rapid laser excitation and the relaxation of the bound dye (5 ps) through vibrational modes to generate heat. For CALI to be based on a photothermal mechanism, the temperature increase at the site of the protein of interest after laser treatment must be sufficiently high to result in thermal denaturation. Photochemical damage would occur through the generation offree radicals by the relaxation ofthe...

Adenomatous Polyposis Coli Is Differentially Distributed in Growth Cones and Modulates Their Steering

“…The micro-CALI setup was built up and used as described previously (Beermann and Jay, 1994;Mack et al, 2000;Wong and Jay, 2000). Micro-CALI ensures spatially limited inactivation (10 m laser spot diameter) of specific proteins/protein domains in cellular subregions via hydroxyl radicals generated by malachite green isothiocyanate (MITC)-coupled antibodies bound to these proteins (Linden et al, 1992;Liao et al, 1994). IgG fractions of antisera APC-m and APC-87, binding to APC regions distant from the microtubule binding domain, and of nonspecific mouse sera (Sigma) were coupled to MITC (MoBiTec, Göttingen, Germany) as described previously (Buchstaller and Jay, 2000).…”

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“…Therefore, we sought a technique for rapid protein inactivation while carrying out live-cell imaging experiments to better understand Golgi dynamics. Chromophores can inactivate proteins upon illumination by producing reactive oxygen species that can generate adducts, break peptide bonds and induce crosslinking (Jay, 1988;Liao et al, 1994;Surrey et al, 1998). Further, with a half maximal reactive distance of approximately 4 nm, the damage can be largely limited to a target protein and its interacting partners (Bulina et al, 2006).…”

Tools and techniques to measure mitophagy using fluorescence microscopy

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