Protein based fluorescent biosensors for small molecule analytes: Development, optimization and use

Carbonic anhydrase (CA) is the receptor molecule for a divalent metal ion biosensor, particularly Zn(II) and Cu(II). Metal sensing is based on the fluorescent enhancement that occurs when fluorescent sulfonamides (dansylamide or Dapoxyl sulfonamide (Dps)) bind to zinc-bound CA. This sensor has been improved by altering the sequence of CA. The Dps affinity was tightened 9-fold over WT CA in K133T/V135A CA. The introduction of a disulfide bond (A23C/L203C) enhanced the stability of CA, essential for incorporating the unstable, but rapid metal equilibrating E117A CA into a sensor. The E117A/A23C/L203C mutant had a zinc affinity of 0.9 +/- 0.2 nM and an enhanced zinc dissociation rate constant. A coupled assay was developed to easily measure the zinc association (2.5 +/- 0.2 x 103 M-1sec -1) and dissociation rate constants and to follow the enhancement of zinc dissociation by small molecules, such as dipicolinic acid.; To develop a ratiometric zinc sensor, CA was fused to the fluorescent proteins EYFP, DsRed or mRFP as a FRET acceptor with the cell permeable fluorophore Dps as the FRET donor. The largest increase (5-fold) in FRET upon the addition of Dps was seen with CA-DsRed, with a KDapp of 14 pM for zinc. Ratiometric fluorescent imaging of the CA-DsRed and E117A CA-DsRed proteins expressed in Escherichia coli demonstrate that CA binds zinc intracellularly. Addition of extracellular zinc or cell permeable chelators causes an increase or decrease, respectively, in the excitation ratio, consistent with changes in the intracellular free zinc concentration. This method shows promise for use as a sensor to measure the intracellular exchangeable zinc concentration.; Lastly, the periplasmic nitrate binding protein NrtA from Synechocystis was investigated for use as the receptor molecule in a nitrate/nitrite sensor. NrtA binds nitrate, nitrite, chromate, arsenate and sulfate with low micromolar affinities. Single cysteine mutants of NrtA, generated for specific fluorophore modification, had altered nitrate affinities. No labeled proteins were discovered that showed changes in fluorescence intensity or anisotropy upon the addition of nitrate, chromate or arsenate, which is in part attributable to the difficulty of removing the free fluorophore from NrtA.