Improved 'optical highlighter' probes derived from Discosoma red fluorescent protein

¹ University of Minnesota

^* To whom correspondence should be addressed. E-mail: robi0386{at}tc.umn.edu.

Submitted on May 7, 2004
Revised on June 18, 2004
Accepted on 5 November 2004

	Abstract

The tetrameric red fluorescent protein, DsRed, undergoes a rapid ^'red^' to ^'green^' color change evoked by short wavelength (<760nm) femtosecond irradiation - a phenomenon that underpins the application of DsRed as an ^'optical highlighter^' probe for tracking live cells, organelles and fusion proteins (Marchant et al. 2001 Nature Biotechnology 19:645-649). This color change results from selective bleaching of the 'mature' red-emitting species of DsRed and an enhancement of emission from the 'immature' green species, likely caused by dequenching of fluorescence resonance energy transfer (FRET) occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, vivid and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for FRET-based probes.

Key Words: GFP, RFP, fluorescent protein, optical imaging, photolabeling