Faculty Collaboration Database

Improved "optical highlighter" probes derived from discosoma red fluorescent protein. Biophys J 2005 Feb;88(2):1444-57

Date

11/24/2004

Pubmed ID

15556986

Pubmed Central ID

PMC1305146

DOI

10.1529/biophysj.104.045617

Scopus ID

2-s2.0-21244452684 (requires institutional sign-in at Scopus site)   11 Citations

Abstract

The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda < 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.

Author List

Robinson LC, Marchant JS

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Cell Line
Humans
Image Enhancement
Kidney
Luminescent Proteins
Microscopy, Confocal
Microscopy, Fluorescence
Microscopy, Fluorescence, Multiphoton
Molecular Probe Techniques
Protein Engineering
Recombinant Proteins
Spectrometry, Fluorescence