Single molecule analysis

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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

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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


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


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

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

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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)


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


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

Composed of a core domain plus ATP and AMP lids Henzler-Wildman

et al.,

Nature , 450,



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

Genetic code expansion

Incorporation of unnatural amino acids : - Broadening the chemical and biological functionalities - Proteins containing UAAs have novel property

Nonsense codon suppression method

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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

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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

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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