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Single-molecule FRET ( smFRET )

Single-molecule FRET ( smFRET ). Determine the FRET efficiencies of biomolecules with a pair of energy donor and acceptor at a single molecule level. Variety of information Conformational changes Biomolecular interactions.

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Single-molecule FRET ( smFRET )

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  1. Single-molecule FRET (smFRET) • Determine the FRETefficiencies of biomolecules with a pair of energy donor and acceptor at a single molecule level • Variety of information • Conformational changes • Biomolecular interactions Understanding the molecular functions, unfolding/refolding process, and structural dynamics of proteins

  2. Major issue in biosciences • Ensemble average • Single molecule-based technologies enabling us to • manipulate and probe individual molecules • Answer many of fundamental biological questions : • - Protein functions : Dynamics and recognition • - Biomolecular interactions • - Biological phenomenon

  3. Single molecule FRET • Replication • Recombination • Transcription • Translation • RNA folding and catalysis • Protein folding and conformational change • Motor proteins • Signal transduction

  4. Measure the extent of non-radiative energy transfer between the two fluorescent dye molecules, donor and acceptor  Intervening distance which can be estimated from the ratio of acceptor intensity to total emission intensity ex) Conformational dynamics of single molecules in real time by tracking FRET changes • Advantages of FRET technique • - A ratiometric method that allows measurement of the internal distance in • the molecular frame with minimized instrumental noise and drift • - Powerful in revealing population distribution of inter-dye distance

  5. FRET- based single molecule analysis

  6. Experimental design • Imaging surface immobilized molecules with the aid of total internal reflection (TIR) microscopy enabling high throughput data sampling • Single-molecule fluorescence dye - Bright ( Extinction coeff. > 50,000 /M/cm; quantumyield > 0.1) - Photostable with minimal photophysical or chemical and aggregation effects - Small and water soluble with sufficient forms of bio-conjugation chemistries

  7. smFRETpair • Large spectral separation between donor and acceptor emissions • Similar quantum yields and detection efficiencies cf) Fluorescent proteins : low stability, photoinduced blinking Quantum dots : large size (>20 nm), lack of a monovalent conjugation scheme • The most popular single-molecule fluorephores : small (< 1nm) organic dye

  8. Enhancing photostability • Molecular oxygen : effective quencher of a dye’s unfavorable triplet state, but a source of a highly reactive species that ultimately causes photo-bleaching • Vitamin E analogue, Trolox, : excellent triplet-state quencher, suppressing blinking and stimulating long-lasting emission of the popular cyanine dyes • The most popular enzymatic oxygen scavenging system: a mix of glucose oxidase (165 U/mL), catalase(2,170 U/ml), b-D-glucose (0.4 % w/w)

  9. Conjugation

  10. Schematics for single-molecule FRET analysis Prism-type Total Internal Reflection Fluorescence (TIRF) microscope ligand

  11. Detection of fluorescence intensities from two dyes • Electron-multiplying charge-coupled device(EM-CCD) cameras • Usual setup : high quantum efficiency(85-95%) in the 450-700nm range, low effective readout noise (<1 electron r.m.s.) even at the fastest readout speed (> 10 MHz), fast vertical shift speed((< 1 us/row) • To achieve adequate signal-to-noise ratio, ~ 100 total photons need to be detected. More than 105 photonscan be collected from single dye molecules before photobleaching, more than 103 data points can be obtained. • FRET efficiency : IA/(IA + ID), IA = acceptor intensity, ID = donor intensity - Provide only an approximate indicator of the inter-dye distance because of uncertainty in the orientation factor between the two fluorophores and the required instrumental corrections - Correction factor : difference in quantum yield and detection efficiency between donor and acceptor

  12. Immobilization of dye-labeled biomolecules on a surface

  13. Sample chamber

  14. Limitations of sm FRET • Attachment of at least two intrinsic dyes to the molecule of interest • Weakly interacting fluorescent species are difficult to study • Insensitive to distance change outside the 2 ~8 nm inter-dye distance • Time resolution is limited by the frame rate of the CCD camera ( in best case = 1 ms) • Absolute distance estimation is challenging because of the dependence of the fluorescence properies and energy transfer on the environment and orientation of the dyes

  15. Intrinsic motions along an enzymatic reaction trajectory • Adnylatekinases : enzymes that maintain the cellular equilibrium concentration of adenylate nucleotides by catalyzing the reversible conversion of ATP and AMP into two ADP molecules • Composed of a core domain plus ATP and AMP lids Henzler-Wildman et al., Nature, 450, 838-850 (2007)

  16. Challenging issue in smFRET • Labeling of proteins with two fluorescent dyes (donor and acceptor) • Most common conventional method for labeling involves: - Introduction of two cysteine residues into desired sites on proteins  Dye heterogeneity  Limited to the nucleic acid-interacting proteins and a subset of proteins that are tolerable to cysteine mutations Site-specific dual-labeling of proteins

  17. Genetic code expansion Met Arg • Incorporation of unnatural amino acids: - Broadening the chemical and biological functionalities - Proteins containing UAAs have novel property • Nonsense codon suppression method • Introduce a stop codon (TAG) at a specific site of a target gene • BioorthogonalaminoacyltRNAsynthetase and tRNA pair for UAA • Expression of protein containing UAA His Ser UAA Translation Site-directed mutagenesis Transcription tRNAsynthetase Nonsense codon tRNA Ribosome TAG AGC mRNA

  18. Site-specific labeling using unnatural amino acid • Dual-labeling of maltose binding protein (MBP) • Incorporation of azido-phenylalanine into Lys42 via an amber codon (TAG) • - Engineered tyrosyl-tRNAsynthetase/tRNACUA of Methanococcusjannaschi • - Conjugation with Cy5-alkyne by click chemistry • Incorporation of cysteine residue into Lys370 Lys42AzF/ Lys370Cys wt Lys42AzF Seoet al., Anal Chem(2011)

  19. Single-molecule FRET measurements Prism-type Total Internal Reflection Fluorescence (TIRF) microscope ligand Time resolution: 50 ms

  20. smFRET analysis of dual-labeled MBP Dual-labeled MBP using UAA Histograms of FRET efficiency Much clearer picture for the folded and unfolded states in smFRET Seoet al., Anal Chem(2011)

  21. Site-specific dual-labeling using two UAAs for smFRET analysis • Incorporation of p-acetylphenylalanine and alkynelysineinto Thr34 and Gly113 on Calmodulin • - Evolution of Methanosarcinamazeipyrrolysyl-tRNAsynthetase(PylRS) for improved • incorporation of AlK: L301M and Y306L • - p-Acetylphenylalanyl-tRNAsynthetase/tRNACUA • Conjugation of two dyes (Cy3-hydrazide and Cy5-azide) via ketone-oxyamineand click reactions Fluorescence scan Calmodulin Lane 1 : CaM Lane 2 : Dual-labeled CaM Alkynelysine (AlK) ρ-acetylphenylalanine (AcF)

  22. Analysis of conformational change by smFRET M13 Ca2+ Histograms of FRET efficiency for M13-induced conformational change of CaM

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