Improved "optical highlighter" probes derived from discosoma red fluorescent protein

Lisbeth C. Robinson, Jonathan S Marchant

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (λ < 760 nm) femtosecond irradiation-a phenomenon that underpins the application of DsRed as an "optical highlighter" probe for tracking live cells, organelles, and fusion proteins. 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 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 acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more 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 fluorescence resonance energy transfer-based probes.

Original languageEnglish (US)
Pages (from-to)1444-1457
Number of pages14
JournalBiophysical journal
Volume88
Issue number2
DOIs
StatePublished - Feb 2005

Bibliographical note

Funding Information:
This work was supported by a CAREER Fellowship Grant from the National Science Foundation (0237946).

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

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