Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Abstract

Fluorescent proteins are increasingly becoming actuators, rather than just sensors, in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage [1]. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is very useful to avoid uncontrolled photodamage. Indeed, fluorescent proteins have been successfully used in CALI, although of the inactivation mechanisms by ROS are dependent on the fluorescent protein used and are not fully understood [2,3]. Here, we present a quantitative study of the ability of TagRFP to produce ROS, in particular singlet oxygen. TagRFP is able to photosensitize singlet oxygen with an estimated quantum yield of 0.004 [4]. This is the first estimation of a quantum yield of singlet oxygen production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime, which reflects relatively high oxygen accessibility to the chromophore compared to EGFP. Our results provide photophysical insight that allows the understanding of the mechanism behind CALI. Moreover, it has implications in improving photobleaching in fluorescent proteins. [1] K. Jacobson, Z. Rajfur, E. Vitriol, K. Hahn, Trends Cell Biol.2008, 18, 443. [2] M. A. McLean, Z. Rajfur, Z. Z. Chen, D. Humphrey, B. Yang, S. G. Sligar, K. Jacobson, Anal. Chem. 2009, 81, 1755. [3] M. E. Bulina et al, Nat .Biotechnol.2006, 24, 95..[4] X. Ragas, L. P. Cooper, J. H. White, S. Nonell, C. Flors, submitted.