Abstract
We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.
Highlights
Two-color fluorescent protein biosensors, expressed in living cells, offer a powerful approach for measuring molecular structural changes by fluorescence resonance energy transfer (FRET) [1]to evaluate biomolecular mechanisms and develop therapeutic approaches [2]
We have found that the expression of genetically-encoded biosensors, fusing GFP and RFP to the target protein, provides the expected advantages of a live-cell assay, because compounds must permeate the cell under truly physiological conditions, and improves the uniformity of the biosensor itself, because it eliminates the heterogeneity introduced by protein purification and labeling with fluorescent dyes [16,17]
The direct waveform recording (DWR) method uses high-energy pulsed lasers in order to acquire thousands of photons emitted from approximately 5000 living cells expressing FRET biosensors in each 5 μL well
Summary
To evaluate biomolecular mechanisms and develop therapeutic approaches [2] This is especially powerful when FRET is measured by a decrease in the donor’s fluorescence lifetime (FLT), increasing the precision of detecting protein structural changes by a factor of 30, compared with the measurement of FRET by intensity [3,4]. The throughput (rate at which successive measurements can be made at equivalent precision) for FLT detection is limited to about 0.1 per second for 1% precision, using the conventional method of time-correlated single-photon counting (TCSPC). The DWR approach has enabled (a) the detection of protein structural changes with high precision during submillisecond biochemical transients [5] and (b) high-throughput screening (HTS) using the first truly high-throughput nanosecond-resolution fluorescence lifetime microplate reader (FLT-PR) [2,6,7]. In the FLT-PR, throughput is limited by the rate at which the plate can be translated, so the effective increase in throughput is ~100-fold, compared with TCSPC
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