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 accep-tor

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 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 read-out 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 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 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 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 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

SerUAA

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