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Structural Basis for Efficient Chromophore Communication and Energy Transfer in a Constructed Didomain Protein Scaffold

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School of Biosciences and School of Chemistry, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, United Kingdom
§ School of Medicine, Cardiff University, UHW Main Building, Heath Park, Cardiff CF14 4XN, United Kingdom
Department of Chemistry, University of Cambridge, Lensfield Road Cambridge, UK CB2 1EW, United Kingdom
EMBL c/o DESY, 22603 Hamburg, Germany
Cite this: J. Am. Chem. Soc. 2012, 134, 33, 13632–13640
Publication Date (Web):July 23, 2012
Copyright © 2012 American Chemical Society

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    The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b562 (cyt b562). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b562 was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H2O2, with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ∼17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.

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    Methods, Table S1, and Figures S1–S11. This material is available free of charge via the Internet at

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    The structure of CG6 has been submitted to the PDB under the code 3U8P.

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