A Multipurpose receptor composed of promiscuous proteins. Analyte detection through pattern recognition.
A multipurpose receptor akin to the "electronic nose" was composed of coumarin-labeled mutants of human glutathione transferase A1. We have previously constructed a kit for site-specific modification of a lysine residue (A216K) using a thiol ester of glutathione (GSC-Cou bio) as a modifying reagent. In the present investigation, we scrambled the hydrophobic binding site (H-site) of the protein scaffold through mutations at position M208 via random mutagenesis and isolated a representative library of 11 A216K/M208X mutants. All of the double mutants could be site-specifically labeled to form the K216 Cou conjugates. The labeled proteins responded to the addition of different analytes with signature changes in their fluorescence spectra resulting in a matrix of 96 data points per analyte. Ligands as diverse as n-valeric acid, fumaric acid monoethyl ester, lithocholic acid, 1-chloro-2,4-dinitrobenzene (CDNB), glutathione (GSH), S-methyl-GSH, S-hexyl-GSH, and GS-DNB all gave rise to signals that potentially can be interpreted through pattern recognition. The measured K d values range from low micromolar to low millimolar. The cysteine residue C112 was used to anchor the coumarin-labeled protein to a PEG-based hydrogel chip in order to develop surface-based biosensing systems. We have thus initiated the development of a multipurpose, artificial receptor composed of an array of promiscuous proteins where detection of the analyte occurs through pattern recognition of fluorescence signals. In this system, many relatively poor binders each contribute to detailed readout in a truly egalitarian fashion.
Viljanen
Johan
J
IFM, Linköping University, S-581 83, Linköping, Sweden.
Larsson
Jenny
J
Larsson
Andréas
A
Broo
Kerstin S
KS
D018360:Crystallography, X-Ray; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D026901:Membrane Transport Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D040081:Protein Array Analysis; D017510:Protein Folding
D026901:Membrane Transport Proteins; C484549:SLC33A1 protein, human