Transmembrane proteins as biosensors

Abstract

The invention relates to engineered transporter proteins comprising at least one fluorescent reporter covalently bound to the transporter protein, wherein the transporter proteins of the present invention comprise a structural inverted repeat motif, with the motif comprising a first and second subunit that are structural inverted repeats of one another and that are joined to one another by a polypeptide loop.

1 . An engineered transporter protein comprising at least one fluorescent reporter covalently bound to the transporter protein, with the transporter protein comprising a structural inverted repeat motif comprising a first and second subunit that are structural inverted repeats of one another and that are joined to one another by a polypeptide loop, wherein the at least one fluorescent reporter is covalently bound to the polypeptide loop. 
     
     
         2 . The engineered transporter protein of  claim 1 , wherein the fluorescent reporter is a fluorescent protein. 
     
     
         3 . The engineered transporter protein of  claim 2 , wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP) and circular permutated green fluorescent protein (cpGFP) 
     
     
         4 . The engineered transporter protein of  claim 1 , further comprising at least one linker peptide that links the fluorescent reporter to the polypeptide loop. 
     
     
         5 . The engineered transporter protein of  claim 4 , comprising two linker peptides that link the fluorescent reporter to the polypeptide loop. 
     
     
         6 . The engineered transporter protein of  claim 5 , wherein each of the first and second subunits of the structural inverted repeats independently comprises three, four, five, six or seven alpha helices. 
     
     
         7 . The engineered transporter protein of  claim 6 , wherein the engineered transporter protein is a member of the AMT/MEP/RH superfamily of transporters. 
     
     
         8 . A nucleic acid encoding the engineered transporter protein of  claim 1 . 
     
     
         9 . A vector comprising the nucleic acid of  claim 8 . 
     
     
         10 . A host cell comprising the vector of  claim 9 . 
     
     
         11 . The host cell of  claim 10  selected from the group consisting of a plant cell and an animal cell. 
     
     
         12 . A method of monitoring transport of a target analyte across a cell membrane, the method comprising inserting the engineered transporter protein of  claim 1  into the cell membrane to control influx and efflux of the target analyte across the membrane and detecting changes in fluorescence of the fluorescent reporter, wherein a change in fluorescence indicates transport of the target analyte across the membrane through the engineered transporter protein. 
     
     
         13 . The method of  claim 12 , wherein the cell is a eukaryotic cell. 
     
     
         14 . The method of  claim 12 , wherein the target analyte is an amino acid.