Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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Single-molecule FRET monitors CLC transporter conformation and subunit independence

10.1101/2020.09.07.286831 ◽

2020 ◽

Author(s):

Ricky C. Cheng ◽

Ayush Krishnamoorti ◽

Vladimir Berka ◽

Ryan J Durham ◽

Vasanthi Jayaraman ◽

...

Keyword(s):

Conformational Change ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Chloride Ions ◽

Conformational State ◽

Triple Mutant ◽

Transport Pathways ◽

Single Molecule Fret ◽

Extracellular Side

Abstract“CLC” transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion access to and from the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Förster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Å) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.SUMMARYCheng, Krishnamoorti et al. use single-molecule Förster energy resonance transfer measurements to monitor the conformation of a CLC transporter and to show that the conformational state is not influenced by the neighboring subunit.

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Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

10.1101/407684 ◽

2018 ◽

Author(s):

Robert B. Quast ◽

Fataneh Fatemi ◽

Michel Kranendonk ◽

Emmanuel Margeat ◽

Gilles Truan

Keyword(s):

Single Molecule ◽

Conformational Equilibrium ◽

Fluorescent Dyes ◽

Photophysical Properties ◽

Accurate Determination ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

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Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

International Journal of Molecular Sciences ◽

10.3390/ijms20163859 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3859 ◽

Cited By ~ 4

Author(s):

Michael Winkler ◽

Florian Wrensch ◽

Pascale Bosch ◽

Maike Knoth ◽

Michael Schindler ◽

...

Keyword(s):

Antiviral Activity ◽

Protein Interactions ◽

Viral Entry ◽

Cellular Localization ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

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Quantitative single molecule FRET efficiencies using TIRF microscopy

Faraday Discussions ◽

10.1039/c5fd00100e ◽

2015 ◽

Vol 184 ◽

pp. 131-142 ◽

Cited By ~ 8

Author(s):

Lasse L. Hildebrandt ◽

Søren Preus ◽

Victoria Birkedal

Keyword(s):

Single Molecule ◽

Structural Information ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Molecular Complexes ◽

Distance Information ◽

Single Molecule Level ◽

Single Molecule Fret ◽

Wide Range ◽

Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

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Affinity of Skp to OmpC revealed by single-molecule detection

Scientific Reports ◽

10.1038/s41598-020-71608-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sichen Pan ◽

Chen Yang ◽

Xin Sheng Zhao

Keyword(s):

Single Molecule ◽

Molecular Chaperones ◽

Outer Membrane Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Correlation Spectroscopy ◽

Hill Coefficient ◽

Stoichiometric Ratio ◽

Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

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Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells

The Journal of Cell Biology ◽

10.1083/jcb.200605097 ◽

2007 ◽

Vol 176 (1) ◽

pp. 51-63 ◽

Cited By ~ 84

Author(s):

Dawen Cai ◽

Adam D. Hoppe ◽

Joel A. Swanson ◽

Kristen J. Verhey

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Structural Organization ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Protein Activity ◽

Kinesin Light Chain ◽

Initial Interaction

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.

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Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex

eLife ◽

10.7554/elife.16886 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 24

Author(s):

Ucheor B Choi ◽

Minglei Zhao ◽

Yunxiang Zhang ◽

Ying Lai ◽

Axel T Brunger

Keyword(s):

Conformational Change ◽

Single Molecule ◽

Neurotransmitter Release ◽

Plasma Membranes ◽

Molecular Mechanisms ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Snare Complex ◽

Spontaneous Release ◽

Trans Conformation

Complexin regulates spontaneous and activates Ca2+-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.

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Live-Cell Imaging Probes to Track Chromatin Modification Dynamics

Microscopy ◽

10.1093/jmicro/dfab030 ◽

2021 ◽

Author(s):

Yuko Sato ◽

Masaru Nakao ◽

Hiroshi Kimura

Keyword(s):

Fluorescent Dyes ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Chromatin Modification ◽

Resonance Energy ◽

Cell Types ◽

Live Cell ◽

Chromatin Modifications ◽

Chromatin Regulation ◽

Single Chain

Abstract The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized both by small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind to specific modifications weakly, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and, differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformation changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation, or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms.

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Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815826116 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4275-4284 ◽

Cited By ~ 12

Author(s):

Stefan Niekamp ◽

Jongmin Sung ◽

Walter Huynh ◽

Gira Bhabha ◽

Ronald D. Vale ◽

...

Keyword(s):

Single Molecule ◽

Open Source Software ◽

Single Molecules ◽

Coiled Coil ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Distance Measurements ◽

Single Molecule Level ◽

Wide Range ◽

Different Color

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil “stalk” of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

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