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DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions
James P Sumner1, Nissa M Westerberg, Andrea K Stoddard
1Department of Chemistry, The University of Michigan, 930N University AVE, Ann Arbor, MI 48109, USA.
Biosensors & Bioelectronics
|July 20, 2005
Summary
Wild type Red fluorescent protein (DsRed) shows high selectivity and sensitivity for copper ions (Cu+ and Cu2+) down to nanomolar levels. This coral-derived protein is significantly more effective than Green Fluorescent protein (GFP) for copper detection.
Area of Science:
- Biochemistry
- Biotechnology
- Environmental Science
Background:
- Red fluorescent protein (DsRed) is an intrinsically fluorescent protein derived from tropical corals.
- Copper ions (Cu+ and Cu2+) are essential micronutrients but toxic at higher concentrations, necessitating sensitive detection methods.
- Existing fluorescent proteins like Green Fluorescent protein (GFP) have limitations in sensitivity and selectivity for metal ion detection.
Purpose of the Study:
- To investigate the potential of wild type DsRed as a highly selective and sensitive biosensor for copper ions.
- To quantify the binding affinity and detection limits of DsRed for both monovalent and divalent copper.
- To compare the performance of DsRed with GFP and its mutants for copper ion sensing.
Main Methods:
- Characterization of DsRed's fluorescence response to varying concentrations of Cu+ and Cu2+.
- Determination of binding constants (K(d)) using single binding isotherms.
- Assessment of selectivity in the presence of other heavy metal ions.
- Computational modeling to predict copper binding sites within the DsRed structure.
Main Results:
- Wild type DsRed exhibits high selectivity and sensitivity for both Cu+ and Cu2+, with nanomolar detection limits.
- The protein maintains selectivity even when other heavy metal ions are present.
- Binding affinities (K(d)) were determined to be 450 nM for Cu+ and 540 nM for Cu2+.
- DsRed's sensitivity to Cu2+ (below 1 ppb) surpasses that of wild type GFP by seven orders of magnitude and engineered GFP mutants by 40 times.
Conclusions:
- Wild type DsRed is a highly effective and sensitive fluorescent biosensor for copper ions.
- DsRed offers superior performance compared to GFP and its mutants for copper detection.
- The findings suggest potential applications for DsRed in environmental monitoring and biological studies requiring precise copper ion quantification.