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
- Biomolecular interactions
- Biological phenomenon
Single molecule FRET
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
- 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
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
 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
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 photons can be collected from single dye
molecules before photobleaching, more than 103 data points can be
 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
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
 Weakly interacting fluorescent species are difficult to study
 Insensitive to distance change outside the 2 ~8 nm inter-dye
 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, 838-850 (2007)
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
 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
tRNA synthetase
Nonsense codon
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/tRNACUA of Methanococcus jannaschi
- Conjugation with Cy5-alkyne by click chemistry
 Incorporation of cysteine residue into Lys370
Lys42AzF Lys370Cys
Seo et al., Anal Chem (2011)
Single-molecule FRET measurements
Prism-type Total Internal Reflection Fluorescence (TIRF) microscope
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/tRNACUA
 Conjugation of two dyes (Cy3-hydrazide and Cy5-azide) via ketone-oxyamine and click reactions
ρ-acetylphenylalanine (AcF)
Fluorescence scan
Lane 1 : CaM
Lane 2 : Dual-labeled CaM
Alkynelysine (AlK)
Analysis of conformational change by smFRET
Histograms of FRET efficiency for M13-induced conformational change of CaM

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