Analysis of ligand binding to a ribose biosensor using site-directed mutagenesis and fluorescence spectroscopy.
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 microM. 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 degrees 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.
Vercillo
Natalie C
NC
Department of Chemistry, University of Richmond, Gottwald Center for Sciences, Richmond, Virginia 23173, USA.
Herald
Kaitlin J
KJ
Fox
John M
JM
Der
Bryan S
BS
Dattelbaum
Jonathan D
JD
D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D015374:Biosensing Techniques; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012266:Ribose / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D033902:Periplasmic Binding Proteins; C028003:RbsB protein, E coli; D012266:Ribose; D000409:Alanine