Phanta: A Non-Fluorescent Photochromic Acceptor for pcFRET

Abstract

We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure‐guided surface engineering

“…In addition, it has been shown that stable or “superfolder” proteins, are also more tolerant to mutagenesis compared with their less stable counterparts, enabling further engineering outcomes that, in some cases, may not have been otherwise possible . In this regard, both eCGP123 and TGP have been used to generate a range of photoswitching and photochromic proteins, (Langan, et al ., 2014, in preparation). High solubility and efficient folding are desirable properties when the FP is used as a fusion partner to prevent non‐specific interactions and aggregation that may create artifacts of subcellular distribution .…”

“…Since chromophore formation and fluorescence is dictated by the protein fold, which is encoded by the amino acid composition, even slight variations in the sequence can lead to dramatic changes in photophysical and biophysical characteristics . Discovery of naturally occurring FP variants in parallel with optimization through random or rational mutagenesis, has led to an enormous number of FP‐based proteins including a palette of colors, photochromic versions, photoswitchable FPs, split FP based molecular reporters, inherently fluorescent biosensors, and highly stable variants …”

X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein

“…GFP-like proteins are valuable tools used extensively in molecular cell biology research [ 1 – 5 ]. In these genetically encoded probes, a tripeptide sequence undergoes a series of post-translational modifications to form a chromophore shielded from bulk solvent within a characteristic 11-stranded β-barrel [ 6 ].…”

“…The fluorescent DsRed (Φ F , 0.70) was converted to the chromoprotein DsRed-NF (Φ F , <0.001) by introducing four amino acid substitutions [ 22 ]. Most recently Phanta, an orange weakly fluorescent photoswitching protein was reported to have utility as a photochromic FRET (pcFRET) acceptor for EGFP, thereby overcoming the requirement for expensive time-resolved fluorescence imaging instrumentation when using non-fluorescent acceptors [ 5 ].…”

Dual observation of the ATP-evoked small GTPase activation and Ca2+ transient in astrocytes using a dark red fluorescent protein

“…This vivid color is most likely due to strong absorption of light by the fluorescent protein in these bacteria. Subsequently, we further examined the identified colonies under blue light to screen the dark colonies in a way similar to a previously reported method1118. As a result, we identified two colonies that had a blue color in daylight but showed dark fluorescence under blue light.…”