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Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

¹ Duke University Medical Center, Department of Biochemistry, Durham, North Carolina 27710, USA
² Wayne State University, Department of Chemistry, Detroit, Michigan 48202, USA
³ University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, Maryland 21201, USA

(RECEIVED September 28, 2004; FINAL REVISION November 8, 2004; ACCEPTED November 9, 2004)

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.

Keywords: biosensors; protein design; periplasmic binding proteins; maltose binding protein; fluorescence; allosteric mechanism

Abbreviations: DTT, 1,4-dithio-D,L-threitol • PBP, bacterial periplasmic binding protein • MBP, Escherichia coli maltose binding protein • IANBD, N-((2-(iodoacetoxy)-ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole • ICT, intramolecular charge transfer • SASA, solvent accessible surface area • , average fluorescence lifetime

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041146005.

Reprint requests to: Homme W. Hellinga, Box 3711, Duke University Medical Center, Department of Biochemistry, Durham, NC 27710, USA; e-mail: hwh{at}biochem.duke.edu; fax: (919) 684-8885.

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