Red fluorescent protein eqFP611 and its genetically engineered dimeric variants

Joerg Wiedenmann; Beatrice Vallone; Fabiana Renzi; Karin Nienhaus; Sergey V. Ivanchenko; Carlheinz Roecker; Gerd Ulrich Nienhaus

doi:10.1117/1.1854680

1 January 2005 Red fluorescent protein eqFP611 and its genetically engineered dimeric variants

Joerg Wiedenmann, Beatrice Vallone, Fabiana Renzi, Karin Nienhaus, Sergey V. Ivanchenko, Carlheinz Roecker, Gerd Ulrich Nienhaus

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Journal of Biomedical Optics, Vol. 10, Issue 1, 014003 (January 2005). https://doi.org/10.1117/1.1854680

Abstract

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four β cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form.

©(2005) Society of Photo-Optical Instrumentation Engineers (SPIE)

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Joerg Wiedenmann, Beatrice Vallone, Fabiana Renzi, Karin Nienhaus, Sergey V. Ivanchenko, Carlheinz Roecker, and Gerd Ulrich Nienhaus "Red fluorescent protein eqFP611 and its genetically engineered dimeric variants," Journal of Biomedical Optics 10(1), 014003 (1 January 2005). https://doi.org/10.1117/1.1854680

Published: 1 January 2005

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