Abstract
In this article, we report for the first time on the copper (Cu
2+) binding characteristics of the far-red fluorescent protein, HcRed, and its application in the development of a reagentless sensing system for copper. The far-red emission of HcRed (λ
max
=
645
nm) where background cellular fluorescence is low should prove to be advantageous in the development of the sensing system. In the studies performed in our laboratory, we found that the fluorescence of HcRed is quenched in the presence of copper ions (Cu
2+). The results obtained through UV-visible and circular dichroism spectra generated in the presence and absence of copper, as well as Stern–Volmer plots at different temperatures, indicate static quenching of HcRed fluorescence in the presence of copper, possibly through the formation of a copper–protein complex. On the basis of this observation, we developed a reagentless sensing system for the detection of copper(II) based on HcRed as the biosensing element. A detection limit for Cu
2+ in the nanomolar range was obtained. HcRed was found to bind copper ions selectively when compared with other divalent ions. A dissociation constant of 3.6
μM was observed for copper binding. Histidine and cysteine residues are commonly involved in copper binding within proteins; therefore, to investigate the role of these amino acids present in HcRed, we chemically modified Cys and His residues using iodoacetamide and diethyl pyrocarbonate, respectively. The effect of copper addition on the fluorescence of the chemically modified HcRed was investigated. The His modification of HcRed substantially affected copper ion binding, pointing to histidine as the possible amino acid residue involved in the binding of copper ions in HcRed. A purification strategy for HcRed was also developed based on a copper immobilized affinity column without the addition of any affinity tag on the protein. The HcRed-based copper sensing system can potentially be employed to perform intracellular copper detection by genetically encoding the biosensing element or can be employed in environmental sensing.
