Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage.

In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.

[1]  Michel Bouvier,et al.  Real-time monitoring of receptor and G-protein interactions in living cells , 2005, Nature Methods.

[2]  K. Tsumoto,et al.  Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay. , 2000, Protein engineering.

[3]  A. Hanyaloglu,et al.  Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells , 2001, Letters in Peptide Science.

[4]  Igor L. Medintz,et al.  Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates , 2006, Nature materials.

[5]  A. Campbell,et al.  Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin. , 2001, The Biochemical journal.

[6]  P. Molinari,et al.  Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells. , 2008, The Biochemical journal.

[7]  H. Fessi,et al.  Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin. , 2004, Thrombosis research.

[8]  Y. Ohmiya.  Basic and Applied Aspects of Color Tuning of Bioluminescence Systems , 2005 .

[9]  J. Tavaré,et al.  Rapid caspase‐3 activation during apoptosis revealed using fluorescence‐resonance energy transfer , 2000, EMBO reports.

[10]  S. Gambhir,et al.  Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. , 2007, Journal of the American Chemical Society.

[11]  W W Ward,et al.  An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. , 1979, The Journal of biological chemistry.

[12]  A. Cox,et al.  An experiment to measure Mie and Rayleigh total scattering cross sections , 2002 .

[13]  J. Chang.  Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. , 1985, European journal of biochemistry.

[14]  Harold A Scheraga,et al.  The thrombin-fibrinogen interaction. , 2004, Biophysical chemistry.

[15]  S. V. Van Doren,et al.  Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect. , 2007, Analytical biochemistry.

[16]  R. Mitra,et al.  Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. , 1996, Gene.

[17]  S. Coughlin,et al.  How the protease thrombin talks to cells. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Blumer,et al.  Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast. , 2002, Methods.

[19]  Bin Zhang.  Design of FRET-based GFP probes for detection of protease inhibitors. , 2004, Biochemical and biophysical research communications.

[20]  K. Eidne,et al.  Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET) , 2006, Nature Methods.

[21]  S. Gambhir,et al.  An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects. , 2007, Cancer research.

[22]  Maarten Merkx,et al.  Enhanced Sensitivity of FRET‐Based Protease Sensors by Redesign of the GFP Dimerization Interface , 2007, Chembiochem : a European journal of chemical biology.

[23]  T. Issad,et al.  The use of resonance energy transfer in high-throughput screening: BRET versus FRET. , 2002, Trends in pharmacological sciences.

[24]  C. A. Parker,et al.  Fluorescence spectrometry. A review , 1962 .

[25]  J. Bockaert,et al.  Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer. , 2008, Biophysical journal.

[26]  K. Tsumoto,et al.  Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer. , 2001, Analytical biochemistry.

[27]  R. Heim,et al.  Using GFP in FRET-based applications. , 1999, Trends in cell biology.

[28]  Kevin Truong,et al.  Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics , 2006, Biotechnology Letters.

[29]  Patrick S Daugherty,et al.  Evolutionary optimization of fluorescent proteins for intracellular FRET , 2005, Nature Biotechnology.

[30]  J. Miller,et al.  Statistics and chemometrics for analytical chemistry , 2005 .

[31]  D. Piston,et al.  Fluorescent protein FRET: the good, the bad and the ugly. , 2007, Trends in biochemical sciences.

[32]  T. Főrster,et al.  10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation , 1959 .

[33]  S. Grant,et al.  A Novel Sensing Technique to Detect Thrombin , 2004 .

[34]  N. Mahajan,et al.  Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis. , 1999, Chemistry & biology.

[35]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[36]  R. Eglen.  Assessing GPCR activation using protein complementation: a novel technique for HTS. , 2007, Biochemical Society transactions.