Minimalist Approaches to Protein Labelling: Getting the Most Fluorescent Bang for Your Steric Buck

2014 ◽  
Vol 67 (5) ◽  
pp. 686 ◽  
Author(s):  
Lee C. Speight ◽  
Moumita Samanta ◽  
E. James Petersson
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Unnatural Amino Acid ◽  
Spectroscopic Techniques ◽  
Environmental Sensitivity ◽  
Protein Labelling ◽  
Single Molecule Level ◽  
Wavelength Absorption

Fluorescence methods allow one to monitor protein conformational changes, protein–protein associations, and proteolysis in real time, at the single molecule level and in living cells. The information gained in such experiments is a function of the spectroscopic techniques used and the strategic placement of fluorophore labels within the protein structure. There is often a trade-off between size and utility for fluorophores, whereby large size can be disruptive to the protein’s fold or function, but valuable characteristics, such as visible wavelength absorption and emission or brightness, require sizable chromophores. Three major types of fluorophore readouts are commonly used: (1) Förster resonance energy transfer (FRET); (2) photoinduced electron transfer (PET); and (3) environmental sensitivity. This review focuses on those probes small enough to be incorporated into proteins during ribosomal translation, which allows the probes to be placed on the interiors of proteins as they are folded during synthesis. The most broadly useful method for doing so is site-specific unnatural amino acid (UAA) mutagenesis. We discuss the use of UAA probes in applications relying on FRET, PET, and environmental sensitivity. We also briefly review other methods of protein labelling and compare their relative merits to UAA mutagenesis. Finally, we discuss small probes that have thus far been used only in synthetic peptides, but which have unusual value and may be candidates for incorporation using UAA methods.

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

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

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.

scholarly journals Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Abhishek Mazumder ◽  
Richard H Ebright ◽  
Achillefs Kapanidis
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Extensive Study ◽  
Active Centre ◽  
Transient Nature ◽  
Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

Identification of Allosteric Fingerprints of Alpha-Synuclein Aggregates in Matrix Metalloprotease-1 and Substrate-Specific Virtual Screening with Single Molecule Insights

2021 ◽  
Author(s):  
Sumaer Kamboj ◽  
Chase Harms ◽  
Derek Wright ◽  
Anthony Nash ◽  
Lokender Kumar ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Single Molecule ◽  
Conformational Changes ◽  
Single Point ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Matrix Metalloproteases ◽  
Single Point Mutation ◽  
Mmp Inhibitor

Abstract Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

scholarly journals Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

2019 ◽  
Vol 116 (17) ◽  
pp. 8350-8359 ◽  
Author(s):  
Jaba Mitra ◽  
Monika A. Makurath ◽  
Thuy T. M. Ngo ◽  
Alice Troitskaia ◽  
Yann R. Chemla ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Spectroscopy ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
Substitution Mutant

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
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.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

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.

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

2019 ◽  
Vol 116 (10) ◽  
pp. 4275-4284 ◽  
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.

scholarly journals Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 49
Author(s):  
Dibyendu Kumar Das ◽  
Uriel Bulow ◽  
Natasha D. Durham ◽  
Ramesh Govindan ◽  
James B. Munro
Keyword(s):  
Membrane Fusion ◽  
Single Molecule ◽  
Conformational Changes ◽  
Ebola Virus ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acidic Ph ◽  
Cellular Environment ◽  
Niemann Pick

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca2+ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment.

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Intensity Changes ◽  
Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

scholarly journals Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Anthony R. Braun ◽  
Elly E. Liao ◽  
Mian Horvath ◽  
Prakriti Kalra ◽  
Karen Acosta ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Time Resolved ◽  
Primary Mouse ◽  
Fret Biosensors

AbstractWe have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

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