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Analysis of ligand binding to a ribose biosensor using site-directed mutagenesis and fluorescence spectroscopy

Department of Chemistry, University of Richmond, Gottwald Center for the Sciences, Richmond, Virginia 23173, USA

(RECEIVED October 2, 2006; FINAL REVISION November 20, 2006; ACCEPTED November 21, 2006)

Computational design of proteins with altered ligand specificity is an emerging method for the creation of new biosensing systems. In this work, we investigated the outcome of site-directed mutagenesis on the Escherichia coli ribose binding protein (RBP), which is frequently used as a design scaffold for computational searches. A ribose biosensor was first constructed whereby an environmentally sensitive fluorescent probe was covalently attached to RBP at position S265C. This protein conjugate displayed a 54% decrease in emission intensity upon the addition of saturating ribose concentrations and exhibited an apparent dissociation constant (K_d) of 3.4 µM. Site-directed mutants within the RBP binding pocket were created and examined for ribose binding ability and overall structural stability. Because as many as 12 mutations are needed to alter ligand specificity in RBP, we measured the effect of single and multiple alanine mutations on stability and signal transduction potential of the ribose biosensor. Single alanine mutations had significant impact on both stability and signaling. Mutations of N190A and F214A each produced melting temperatures >8°C below those observed for the wild-type protein. Residue Q235, located in the hinge region of RBP, appeared to be a hot spot for global protein stability as well. Additional single alanine mutations demonstrated as much as 200-fold increase in apparent K_d but retained overall protein stability. The data collected from this study may be incorporated into design algorithms to help create more stable biosensors and optimize signal transduction properties for a variety of important analytes.

Keywords: biosensor; periplasmic binding proteins; ribose binding protein; fluorescence

Abbreviations: PBP, bacterial periplasmic binding protein; RBP, Escherichia coli ribose binding protein; ABD-F, [4-Fluoro-7-aminosulfonylbenzofurazan]; CD, circular dichroism.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062595707.

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