Dimeric variants of the red fluorescent protein eqFP611 generated by site-directed mutagenesis

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

doi:10.1117/12.529370

14 June 2004 Dimeric variants of the red fluorescent protein eqFP611 generated by site-directed mutagenesis

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

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Proceedings Volume 5329, Genetically Engineered and Optical Probes for Biomedical Applications II; (2004) https://doi.org/10.1117/12.529370
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

Abstract

The red fluorescent protein eqFP611 shows favorable properties for applications as molecular marker. Its usefulness is, however, limited by its tendency to form tetramers at physiological concentrations. To provide a basis for the rational design of monomeric variants, we examined the monomer interfaces in the x-ray structure of eqFP611. The arrangement of the four ß cans is very similar to that of other GFP-like proteins such as DsRed and RTMS5. In eqFP611, the monomers are linked by comparatively weak interactions, as inferred from the dissociation into monomers in the presence of SDS or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. Some structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. Functional dimeric variants could be generated by altering the A/B interface by single point mutations (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface resulted as yet in essentially complete loss of fluorescence. Presumably, the folding of eqFP611 into its functional form relies on A/C interfacial interactions.

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Joerg Wiedenmann, Beatrice Vallone, Fabrizio Renzi, Karin Nienhaus, Sergey V. Ivanchenko, Carlheinz Roecker, and Gerd Ulrich Nienhaus "Dimeric variants of the red fluorescent protein eqFP611 generated by site-directed mutagenesis", Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); https://doi.org/10.1117/12.529370

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