A New-Generation Fluorescent-Based Metal Sensor – iLOV Protein

Abstract

The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a lightoxygen- voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu(2+) was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 micrometer. In addition, Cu(2+)-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu(2+) binding with the iLOV protein.

Live‐Cell Copper‐Induced Fluorescence Quenching of the Flavin‐Binding Fluorescent Protein CreiLOV

“…To determine if the Cu II ‐induced quenching was reversible, we added an equivalent amount of the metal chelator EDTA, and found that the fluorescence was restored to ≈85 % of the original fluorescence and was not further increased with additional chelator (Figure A, B). These results are similar to those previously observed for iLOV . Given that zinc and copper are two of the tightest‐binding biologically relevant metal ions, we also checked the reversibility of the modest Zn II ‐induced quenching.…”

“…We set out to determine how the properties of CreiLOV are affected by the presence of biologically relevant metal ions. Previous work on iLOV demonstrated an inherent affinity for and quenching effects from the presence of Cu II , but CreiLOV has a higher quantum yield than iLOV and is more photostable . Furthermore, although the amino acids that interact with FMN are the same between the two proteins, the proposed metal‐binding amino acids of iLOV are two asparagine residues.…”

“…Genetically encoded metal ion sensors that are not derived from GFP family proteins are rarely found, including those based on single fluorescent proteins . Recently, Ravikumar and co‐workers reported that the flavin‐based fluorescent protein iLOV binds Cu II ( K d =4.72 μ m at pH 7.4) . The same group also reported binding of FbFP variants to arsenic(III) and mercury(II) .…”

LOV-based reporters for fluorescence imaging

“…For instance, iLOV fluorescence is quenched in the presence of copper at physiological pH and is reversible in the presence of the metal ion chelator EDTA [35]. Two asparagine residues within the vicinity of the FMN fluorophore at positions 401 and 425 (based on the amino acid positions in Arabidopsis phototropin 2 from which iLOV was derived) are proposed to coordinate copper binding.…”

Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

“…The site‐specific incorporation of tyrosine analogues not only yielded a pH sensor, but also system that selectively detects Mn(III) . Additionally, it was found that Cu(II) binds to iLOV with a K d of 4.7 µM and quenches its fluorescence selectively and reversibly . Unfortunately, neither of these sensors has been tested in live cell microscopy.…”