title: 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.
authors.affiliation: School of Biotechnology, Department of Biochemistry, Yeungnam University, Gyeongsan 712-749, Republic of Korea.
authors.forename: Yuvaraj
authors.initials: Y
authors.lastname: Ravikumar
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Saravanan Prabhu
authors.initials: SP
authors.lastname: Nadarajan
authors.affiliation: School of Biotechnology, Department of Biochemistry, Yeungnam University, Gyeongsan 712-749, Republic of Korea.
authors.forename: Chong-Soon
authors.initials: CS
authors.lastname: Lee
authors.affiliation: Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea.
authors.forename: Jin-Kyu
authors.initials: JK
authors.lastname: Rhee
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Hyung-Don
authors.initials: HD
authors.lastname: Yun
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D003300:Copper; D004492:Edetic Acid; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals; D010786:Photoreceptor Cells / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008670:Metals; D011506:Proteins; D011994:Recombinant Proteins; D003300:Copper; D004492:Edetic Acid