Fö rster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra-and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.

19 Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to 20 profile intra-and inter-molecular interactions. Through fusion of genetically encoded 21 fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, 22 receptor activation, and protein translocation among other biophysical phenomena.23 Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have 24 been developed. These proteins offer much improved physical properties compared to 25 previous generations of monomeric red FPs that should help facilitate more general 26 adoption of Green/Red FRET. Here we assess the ability of these two proteins, along 27 with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP 28 mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging 29 Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a 30 stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We 31 then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP 32 tandem construct. With these constructs we found that mScarlet-I and mCherry are able 33 to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, 34 mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these 35 same assays. We explore possible explanations for this poor performance but are 36 unable to definitively determine the cause, all though protein maturation seems to play a 37 role. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry 38 and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with mScarlet-I out 39 performing mCherry due it's higher brightness. 40 41 Introduction 42 Genetically encoded Fluorescent Proteins (FPs) have advanced basic and translational 43 biology immensely. Starting with the cloning of the Aequorea victoria green FP[1], a 44 massive and continual effort to expand the number of available FPs with a different 45 physical and spectral properties began. Currently, there is an enormous variety of FPs 46 at all parts of the visible spectrum, and even some parts of the ultraviolet and infrared 47 spectrums. These new proteins were either cloned from other organisms[2-4], or48 developed through evolution of already identified FPs [4][5][6][7][8][9][10][11]. This ever expanding 49 catalog of FPs has been reviewed by others [12][13][14], and new efforts and archives have 50 been created to organize this information, such as the FPbase database [15]. 51As the catalogue of FPs has expanded, so have the potential uses. Of particular 52 note is the use of FPs as biosensors which can measure signaling events, cell 53 metabolites, pH, voltage and more [16]. Many of these biosensors employ Förster 54 Resonance Energy Transfer (FRET) as part of their reporting mechanism, producing a 55 change in acceptor emission upon donor excitation when the quantity of interest 56 changes. FRET is also orientation and distance dep...

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Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by decreases in extracellular pH. The intracellular N and C terminal tails of ASIC1 influence channel gating, trafficking, and signaling in ischemic cell death. Despite several x-ray and cryo-EM structures of the extracellular and transmembrane segments of ASIC1, these important intracellular tails remain unresolved. Here we describe the coarse topography of the chicken ASIC1 intracellular domains determined by FRET, measured using either fluorescent lifetime imaging or patch clamp fluorometry. We find the C terminal tail projects into the cytosol by approximately 35 Å and that the N and C tail from the same subunits are closer than adjacent subunits. Using pH-insensitive fluorescent proteins, we fail to detect any relative movement between the N and C tails upon extracellular acidification but do observe axial motions of the membrane proximal segments towards the plasma membrane. Taken together, our study furnishes a coarse topographic map of the ASIC intracellular domains while providing directionality and context to intracellular conformational changes induced by extracellular acidification.

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors with widespread expression in the central nervous system. Unlike many GPCRs, mGluRs exist and function as obligate, disulfide linked dimers. While all mGluRs can form homodimers, and many can heterodimerize, we have recently published a paper demonstrating that while mGluR1 and 5 may reside in close proximity to each other, and can interact functionally, this interaction does not appear to be due to canonical heterodimerization. Pharmacological evidence supporting this conclusion is largely pharmacological. To ask whether the observed interaction was due to a physical interaction between receptors rather than integration of signaling effects, mutations in each receptor were introduced that uncouple the receptors from G protein signaling, presumably without affecting ligand binding or associated conformational changes. We found that mGluR1 co‐expressed with a double point mutant of mGluR5, Y64A F768D (which right‐shifted the glutamate response and uncouple from G proteins, respectively), the glutamate response of mGluR1 was right shifted. When mGluR1Y74A, a mutant with a 300 fold right‐shifted response, was expressed with mGluR5 F768D, the response was left shifted. Expression of the signaling dead mGluR5 F768D with mGluR1 also altered the pharmacological sensitivity profile, causing mGluR1 to lose sensitivity to 3‐MATIDA and gain sensitivity to MPEP. Together, these findings support the hypothesis that allosteric interactions between the receptors are responsible for the observed functional interactions. Support or Funding Information Supported by an institutional Drug Targets and Mechanisms pilot award.