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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/refold-ing 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
• 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 be-tween the two fluorescent dye molecules, donor and ac-ceptor
Intervening distance which can be estimated from the ra-tio 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 dis-tance
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
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 mono-valent
conjugation scheme
The most popular single-molecule fluorephores : small (< 1nm) organic dye
smFRET pair
Enhancing photostability
Molecular oxygen : effective quencher of a dye’s unfavor-able 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-last-ing 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) mi-croscope
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 105 photons can be collected from single dye molecules before photobleaching, more than 103 data points can be ob-tained.
FRET efficiency : IA/(IA + ID),
IA = acceptor intensity, ID = donor intensity
- Provide only an approximate indicator of the inter-dye distance be-cause
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 equilib-rium 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 accep-
tor)
Most common conventional method for labeling involves:
- Introduction of two cysteine residues into desired sites on pro-
teins 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 pro-teins
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
Met
Arg
His
Ser
UAA
AGC TAG
mRNA
Ribosome
Site-directed mutagenesis
Transcription Translation
Nonsense codon
tRNA
tRNA synthetase
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
Seo et al., Anal Chem (2011)
wt Lys42AzFLys42AzF/Lys370Cys
Single-molecule FRET measurements
Prism-type Total Internal Reflection Fluorescence (TIRF) microscope
ligand
Time resolution: 50 ms
smFRET analysis of dual-labeled MBP
Histograms of FRET effi-ciency
Dual-labeled MBP using UAA
Seo et al., Anal Chem (2011)
Much clearer picture for the folded and unfolded states
in smFRET
Site-specific dual-labeling using two UAAs for smFRET analysis
H2NCOOH
O
COOHH2N
HN
O
O
Alkynelysine (AlK)
H2NCOOH
O
COOHH2N
HN
O
O
ρ-acetylphenylalanine (AcF)
Calmodulin Fluorescence scan
Lane 1 : CaMLane 2 : Dual-labeled CaM
Incorporation of p-acetylphenylalanine and alkynelysine into Thr34 and Gly113 on
Calmodulin - Evolution of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) for im-proved
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
Analysis of conformational change by smFRET
Ca2+
M13
Histograms of FRET efficiency for M13-induced conformational change of CaM