Abstract

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 ± 0.03 to 2.78 ± 0.05 ns for the Y145H mutant and to 2.74 ± 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.

Original languageEnglish (US)
Pages (from-to)13377-13384
Number of pages8
JournalBiochemistry
Volume44
Issue number40
DOIs
StatePublished - Oct 11 2005

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Green Fluorescent Proteins
Fluorescence
Optical Imaging
Imaging techniques
Mutation
Histidine
Cysteine
Fluorophores
Polymerase Chain Reaction
Binary mixtures
Conformations
Screening
Proteins
Detectors
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Development and characterization of green fluorescent protein mutants with altered lifetimes. / Scruggs, Allan W.; Flores, Carole L.; Wachter, Rebekka; Woodbury, Neal W.

In: Biochemistry, Vol. 44, No. 40, 11.10.2005, p. 13377-13384.

Research output: Contribution to journalArticle

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