Transcript Single molecule analysis
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 Understanding the molecular functions, unfolding/refolding process, and structural dynamics of proteins
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
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Biomolecular interactions - Biological phenomenon
Single molecule FRET
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Replication Recombination Transcription Translation RNA folding and catalysis Protein folding and conformational change Motor proteins Signal transduction
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
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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
FRET- based single molecule analysis
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; quantum yield > 0.1) - Photostable with minimal photophysical or chemical and aggregation effects - Small and water soluble with sufficient forms of bio-conjugation chemistries
smFRET pair
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
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)
Conjugation
Schematics for single-molecule FRET analysis
Prism-type Total Internal Reflection Fluorescence (TIRF) microscope ligand
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 10 5 photons can be collected from single dye molecules before photobleaching, more than 10 3 data points can be obtained.
FRET efficiency : I I A A /(I A + I D ), = acceptor intensity, I D = donor intensity
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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
Immobilization of dye-labeled biomolecules on a surface
Sample chamber
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
Intrinsic motions along an enzymatic reaction trajectory
Adnylate kinases : enzymes that maintain the cellular equilibrium concentration of adenylate nucleotides by catalyzing the reversible conversion of ATP and AMP into two ADP molecules
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Composed of a core domain plus ATP and AMP lids Henzler-Wildman
et al.,
Nature , 450,
838-850
(2007)
Challenging issue in smFRET
Labeling of proteins with two fluorescent dyes (donor and acceptor)
Most common conventional method for labeling involves:
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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
Genetic code expansion
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 Bioorthogonal aminoacyl tRNA synthetase and tRNA pair for UAA Expression of protein containing UAA AGC Site-directed mutagenesis TAG tRNA synthetase tRNA Transcription mRNA Nonsense codon Ribosome Arg Met His Ser Translation UAA
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-tRNA synthetase/tRNA CUA of Conjugation with Cy5-alkyne by click chemistry Methanococcus jannaschi Incorporation of cysteine residue into Lys370 wt Lys42AzF Lys42AzF/ Lys370Cys Seo et al ., Anal Chem (2011)
S ingle-molecule FRET measurements
Prism-type Total Internal Reflection Fluorescence (TIRF) microscope ligand Time resolution: 50 ms
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 Seo et al ., Anal Chem (2011)
Site-specific dual-labeling using two UAAs for smFRET analysis
Incorporation of p -acetylphenylalanine and alkynelysine into Thr34 and Gly113 on Calmodulin - Evolution of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) for improved incorporation of AlK : L301M and Y306L p-Acetylphenylalanyl-tRNA synthetase/tRNA CUA Conjugation of two dyes (Cy3-hydrazide and Cy5-azide) via ketone-oxyamine and click reactions Calmodulin ρ-acetylphenylalanine (AcF) Fluorescence scan Lane 1 : CaM Lane 2 : Dual-labeled CaM Alkynelysine (AlK)
Analysis of conformational change by smFRET
M13 Ca 2+ Histograms of FRET efficiency for M13-induced conformational change of CaM