title: Design of a highly specific and noninvasive biosensor suitable for real-time in vivo imaging of mercury (II) uptake.
abstract: Mercury is a ubiquitous pollutant that when absorbed is extremely toxic to a wide variety of biochemical processes. Mercury (II) is a strong, "invisible" poison that is rapidly absorbed by tissues of the intestinal tract, kidneys, and liver upon ingestion. In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. To our knowledge, this engineered protein is a first example of a biosensor that allows for noninvasive and real-time imaging of mercury uptake in a living cell. A major advantage is that its expression can be genetically controlled in many organisms to enable unprecedented studies of tissue specific mercury uptake.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California 93106-9510, USA.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Chapleau
authors.forename: Rebecca
authors.initials: R
authors.lastname: Blomberg
authors.forename: Peter C
authors.initials: PC
authors.lastname: Ford
authors.forename: Martin
authors.initials: M
authors.lastname: Sagermann
mesh_terms: D000042:Absorption; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008628:Mercury / Q000378:metabolism*; D008958:Models, Molecular; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence
chemical_list: C485184:enhanced green fluorescent protein; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D008628:Mercury