Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

Sergei Pletnev,Nadya G Gurskaya,Nadya V Pletneva,Konstantin A Lukyanov,Dmitri M Chudakov,Vladimir I Martynov,Vladimir O Popov,Mikhail V Kovalchuk,Alexander Wlodawer,Zbigniew Dauter,Vladimir Pletnev

doi:10.1074/jbc.m109.054973

Abstract

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

Highlights

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold
We have focused our attention on the distinctive structural features that might be responsible for the observed photophysical properties of the protein
Monomer Structure—The principal structural fold of KillerRed, shared with all members of the green fluorescent protein (GFP) family, is an 11-stranded ␤-barrel having loop caps from both sides and a chromophore embedded in the middle of an internal ␣-helix located on the ␤-barrel axis

Summary

Introduction

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. The first two replacements are the most essential, because they provide a significant contribution to stabilization of the fluorescent cis form of the chromophore by hydrogenbonding the hydroxyl of Tyr66 to the side chains of Asn145 and via a water molecule to Thr201.

Results

Conclusion