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Strukturanalyse > Single molecule FRET > Flashcards

Single molecule FRET Flashcards

1
Q

FRET

A

Förster Resonance Energy Transfer
- it is a process, by which an excited fluorophore (called donor) relaxes to the ground state providing the energy to another fluorophore (called acceptor) for excitation
- the energy is transferred by dipole-dipole coupling (direct electrostatic interaction) if fluorescence transitions in the donor and absorption transitions in the acceptor have the same energy difference (hence are resonant)
- the process does not involve the emission and absorption of a photon, it is radiation less (no wave propagation)

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2
Q

Transfer efficiency (Et)

A
  • ratio of transferred quanta to absorbed quanta
  • can be measured
  • “high” FRET -> Et high
  • “low” FRET -> Et low
  • 0.25 - 1.5 is the best range for the Förster radius (outside of this range the slope isnÄt really usable)
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3
Q

Rotational averaging

A
  • Dynamic mean (Both dipoles rotate fast compared to all relaxation rates): <κ2>=2/3</κ2>
  • One dipole is rotating fast, the other one slowly: κ2≈2/3
  • Static ensemble average, slow rotation: κ2=0,476
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4
Q

Donor in triplet state

A

No adsorption, therefore no emission: donor and acceptor become dark

-> just reducing the effective measurement time

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5
Q

Acceptor in triplet state

A

Everything can happen:
- If no FRET is occuring: donor fluorescence is increasing
- Energy transfer to T1 - TN transition of acceptor: Depending on efficiency of this transfer compared to „standard FRET“ S0 - S1 fluorescence of donor can increase, decrease, or remain constant

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6
Q

Singlet triplet annihilation (STA)

A

Förster-type energy transfer to T1-TN transition

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7
Q

Förster radius

A
  • distance, where transfer rate equals sum of all other rates

-> assumption: kappa^2, J, n are constant

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8
Q

Fluorescent labels

A
  • synthetic dyes: electrons in conjugate pi-systems (pi-electrons from the double bond are essentially free to move in the system)
  • fluorescent proteins: electrons in conjugate pi-systems, strong photo-dynamics, not very stable
  • quantum dots: semiconductor nano-crystals in a box -> the larger the box the lower the energies
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9
Q

Rhodamines

A
  • High quantum yield
  • photo stable
  • vlow intersystem crossing rate
  • Absorption: 488 …. 600 (side groups)
  • BSP.: Fleurescein, Rhodamine 6G, Alexa Fluor
    -> side groups improve solubility in water
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10
Q

Cyanines

A
  • High quantum yield
  • photo stable
  • low intersystem crossing rate
  • but: photo isomerization leading to a dark state

Cy3: Absorption = 520 nm
Cy5: Absorption = 650 nm (da all-trans -> fluorescent, cis -> dark)

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11
Q

NH coupling

A
  • typically with NHS-Ester
  • unspecific labelling (terminal and side chain NH)

-> the terminal NH chain has a different pK-value -> this enables to specifically label the terminal NH group

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12
Q

SH coupling

A
  • typically with maleimide
  • specific labelling of cysteine residues (introduced by mutations, typically in loops regions)

Maleimide -> Thioether

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13
Q

Immobilized molecules

A
  • Long observation times: all dynamics time scales
  • Immobilization can cause artifacts
  • Confocal or wide field/TIRF detection
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14
Q

Gel immobilization

A
  • Entrapment of molecules in gel pores (agarose (low melting otherwise the proteins are destroyed), PAA)
  • Small molecules (e.g. substrates) can still diffuse

-> acrylamide might quench the fluorescent

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15
Q

Immobilization in liposomes

A
  • Entrapment in liposomes smaller than focus size (typ. 50-100 nm)
  • Chemical milieu is fixed (lipid bilayer is sort of a barrier)
  • immobilization of membrane proteins
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16
Q

Immobilizaztion at surfaces

A
  • His Tag, Strep Tag -> Nickel-affinity column for purification
  • Surface modification:
    -> Silanisation (e.g. with Ni-chelator for His-Tag)
    .. protein needs to like glass surfaces
    -> Biotin-labelled PEG or BSA for strep tag (Biotynilation of the protein)
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17
Q

Comparison: Correlation function vs State analysis

A
  • Correlation function does not need threshold
  • Interpretation of correlation function requires model
  • State analysis is easier to understand
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18
Q

Experiments in solution

A
  • free diffusion (Brownsche Molekularbewegung) and confocal detection: every molecule only for short time “visible”
  • internal dynamics accessible in limited time window
  • close to physiological conditions
    -> in the “diffusion experiment” a bunch is called a “burst”
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19
Q

Burst analysis

A

Fluorescence burst: All photons from a molecule during focal transit

-> one burst = one molecule
-> integrative characterization of all photons of one burst each

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20
Q

Histogram

A
  • Histogram over fluorescence parameters of every burst
  • Shot noise leads to broadening of the histograms, even if all bursts have the same fluorescence parameter
  • If different states with different fluorescence parameters are present in the sample: more peaks
  • if state is changing during focal transit: peaks are melting into each other

-> slow exchange: time scale of state kinetics are longer than focal transit = 100 µs
-> fast exchange: kinetics shorter than focal transit
CLOSED: short distance = high E
OPEN: long distance = low E

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21
Q

Correlation analysis: FCS (Fluorescence Correlation Spectroscopy)

A

Diffusion(al dynamics) leads to fluorescence fluctuations, with correlation function

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22
Q

2 Color cross correlation

A
  • Indicates binding (correlated motion) by correlated fluctuations
  • No binding: uncorrelated motion, uncorrelated fluctuations
23
Q

Internal dynamics

A

All processes faster than diffusion (e.g. triplet, FRET internal dynamics…) remain visible

-> changes between conformational states

24
Q

Multi parameter detection

A
  • Purpose: make FRET data more reliable, avoid misinterpretations
    -> detect, record and analyze different fluorescence properties simultaneously in one experiment
  • Important fluorescence parameters related to FRET:
    -> Photon flux (“intensity”)
    -> Fluorescnece lifetime
    -> Anisotropy
    -> Emission spectrum
    -> Excitation spectrum
25
Q

Stoichiometry/FRET

A
  • Stochastic labeling: donor/donor and acceptor/acceptor proteins
  • Selective, alternating excitation of donor / acceptor with two lasers:ALEX (Alternating Laser Excitation) or. PIE (Pulsed Interleaved Excitation)
  • Definition of new quantitity S: Stoichiometry (cf. E)
  • It is in principle sufficient to pulse only the acceptor laser
26
Q

Anisotropy/lifetime

A
  • Perrin equation connects lifetime with anisotropy
  • FRET increases fluorescence anisotropy!
  • Roatational frustration can become visible
  • Attention: short donor lifetimes mean a low number of donor photons!
27
Q

Triplet modification

A
  • Heavy atoms: strong spin orbit coupling, enhance intersystem crossing (fwd and backwd)
  • Triplet quencher (electron exchange) -> shorten triplet lifetime via electron exchange -> contact of electron wave functions is needed!
28
Q

Radical ions - ROXS

A
  • Radical ionic states - long lived dark states
  • Reducing Oxidizing System (ROXS) depopulates those states
29
Q

Energy levels and transitions

A
  • a thorough understanding of the energy levels and transitions in a fluorophore (molecule, that can fluoresce) are key for understanding
    -> the reason why we cannot increase the fluorescence signal to arbitrary
    -> the fluctuations inherent to the fluorescence signal
    -> the ways to count the number of fluorophores
30
Q

Jablonski diagram

A

Energy levels of a fluorophore are visualized in a Jablonski diagram showing
- electronic ground state and excited states (singlet and triplet) as thick lines
- corresponding vibrational states as thin lines
- transitions as arrows
-> vertical arrows: associated with energy uptake/release either by radiation absorption/emission (straight arrows) or heat dissipation by vibrational energy
-> spin flip: Intersystem crossing between singlet (antiparallel spins, total spin S=0, magnetic spin number ms=0) and triplet (parallel spins, total spin S=1, magnetic spin number ms=-1,0,1) system

31
Q

Kasha’s rule

A

irrespective of the absorption transition, the molecule rapidly relaxes via vibrational relaxation or a combination of internal conversion with vibrational relaxation to the vibrational ground state of S1 called S1,0

32
Q

Stokes shift

A
  • emission red shifted with respect to excitation
  • there is some confusion in the literature about the stokes shift: if the absorption led to an excitation of a higher vibrational state, the red shift is also due to vibrational relaxation
  • however, the stokes shift in the strict sense is the red shift of the 0-0 transition of emission (S1,0 -> S0,0) vs the 0-0 transition of absorption (S0,0->S1,0)
    -> in the Jablonski diagram, however, there is no energy difference between these transitions!
  • the reason for this stokes sift is solvent relaxation: the change of the electronic wave functions upon electronic excitation/relaxation leads to an energetically unfavorable orientation of the solvent molecules forcing them to reorient, which leads to an energy difference between the absorption/emission 0-0 transitions
33
Q

Dynamic solution

A

We now ask the question how the occupancy of the first excited state evolves as a function of time with the initial condition of the system being in the ground state
- the reason for this is the following: If we detect a photon, we know that in this moment the molecule is in the ground state

34
Q

Background can be reduced by…

A
  • optimize excitation wavelength (high absorption)
  • use the best (and appropriate) filters!
  • use temporal filtering (pulsed excitation, since fluorescence is delayed in contrast to scattering)
  • use detectors with low dark count rate
35
Q

Rotation of a dipole

A

In general, r can take values between +1 and -1/2 however, if the dipoles are randomly aligned, only values between 0 (complete depolarization, fast rotation) and 0.4 (no rotation of the dipole at all) are possible

36
Q

Rotation in a cone

A

If we now think of a dye coupled to a larger protein, this coupling may lead to a limitation of the mobility, which can be described by the rotation in a cone model

37
Q

Transient sticking

A

It could also happen that the dye is (transiently or permanently) sticking to the protein
- This is typically a result of the dye’s hydrophobicity, which leads to sticking tendency towards hydrophobic patches at the protein surface
- Holds inly if sticking time is longer than excited state lifetime
- The main difference to the rotation in a cone is that this sticking leads to a heterogeneity, i.e. two populations of molecules, which can be a problem for single-molecule experiments

38
Q

Singlet triplet annihilation (STA)

A

An acceptor in the triplet state still may be able to accept energy, via a FRET like dipole-dipole coupling
1. The donor fluorescence can decrease, if STA is more efficient than FRET
2. The donor fluorescence can stay constant, if STA is as efficient as FRET
3. the donor fluorescence can increase, if STA is less efficient than FREZ or not happening at all

39
Q

Amino group labeling

A

typically via NHS ester, unspecifically at lysines or specifically at the N-terminus (depending on pH)

40
Q

Thiol group labeling

A

Maeleimide, specifically label Cysteine

41
Q

Unnatural amino acids labeling

A

Step 1: Incorporation of non-canonical AA
- amber suppression
- - orthogonal tRNA/synthetase pair: essential
– no cross-reaction with the native host translation machinery
– non-sense TAG codon (Amber) becomes a sense codon, incorporating a non-canonical AA

Step 2: Coupling a fluorophore to the non-canonical Amino Acid
- employing the “Click-Chemistry”

42
Q

Experiments with immobilized molecules

A
  • measurements on immobilized molecules allow for observation of single molecules for extended periods in time
  • thus, dynamic heterogeneity is accessible
43
Q

Immobilization in GEL

A
  • the pores of the gel have to be small enough to prevent motion of the protein
  • small ligands can still diffuse through the gel
44
Q

Immobilization in LIPOSOMES

A
  • liposomes can be immobilized by incorporation of functionalized lipids, e.g. biotynilated or PEGylated lipids
  • the diameter of the liposome has to be smaller than the confocal spot diameter of about 200 nm, so that diffusion within the liposome is invisible
45
Q

Immobilization directly on SURFACES

A
  • glass surfaces can be functionalized by silanes with functional groups, e.g. Nickel chelating groups, so that His-tagged proteins can be tethered
  • there is also a range of PEG-coupled antibodies against special tags an the protein available (His-Tag, Strep-Tag, etc)
46
Q

Experiments in solution

A
  • In these experiments, the molecules of interest are dissolved at very low concentrations (sub nano molar range) in a solvent (for proteins typically buffer solutions), where they diffuse freely, i.e., follow Brownian motion
  • fluorescent excitation and detection occurs in the confocal volume of a confocal microscope
  • the confocal volume is therefore placed in drop (some microliters) of the solution with the molecules of interest
  • Brownian motion leads to a fluctuating signal: every time a molecule enters the confocal volume, fluorescence is detected, which is called a fluorescence burst
  • because of the rather sparsely occurring fluorescent bursts and the need of a time resolution down to nanoseconds, the data here are typically accquired as single photon data
47
Q

Burst analysis

A
  • in burst analysis, the first step is the identification of bursts, typically based on a threshold criterion
  • Here, the threshold may concern the total fluorescence signal, the donor signal, the acceptor signal or any combination there of
  • for every burst, the collected photons are analyzed with respect to certain property of fluorescence, e.g. FRET efficiency, fluorescence lifetime, anisotropy and so on:
  • due to shot noise, even if the e.g. FRET efficiencies for one sort of molecules are identical, the distributions visible in the histogram always have a finite width
  • the smaller the number of photons in a burst, the broader the shot-noise limited peak width
48
Q

The influence of FRET dynamics

A
  • depending on the time scale of prospective FRET state interconversions, the peaks in the histogram may melt into each other
  • If the state interconversion is much faster than diffusion, i.e., if many interconversions take place while the molecule is in the focus, only one peak at the population weighted average remains
  • to test, if one peak is present because there is just one FRET state present, or because of fast averaging, correlation analysis (next section) can be used
49
Q

FRET Multi parameter detection and burst analysis

A
  • The term multi parameter detection refers to all methods where more than just one parameter is derived from the photons of a fluorescence burst
  • The bursts can be analyzed with respect to:
    -> their wavelength (e.g. donor or acceptor)
    -> the wavelength that was used for excitation (which fluorophore did absorb?)
    -> the arrival time after pulsed excitation (providing information on the fluorescence lifetime)
    -> the polarization
  • the multi parametric detection greatly enhances the reliability of FRET experiments
  • to visualize the multi parametric experiment, two-dimensional histograms are used
  • here we will mainly refer to solution experiments and burst analysis
50
Q

ALEX (Alternating laser excitation)

A

microsecond switching

51
Q

PIE (pulsed interleaved excitation)

A

picosecond pulses are alternatively applied

52
Q

Triplet quenching

A
  • It would be advantageous to be able to reduce the rate if intersystem crossing and to enhance the rate of reverse intersystem crossing, the latter termed triplet quenching
  • Heavy atoms like Iodine enhance both rates by facilitating spin reversal via spin orbit coupling
  • a reduction of the intersystem crossing rate would require the reduction of the spin orbit coupling, which is mostly not possible
  • we remain with triplet quenching, which is possible by exchanging an electron of reverse spin with another molecule
  • two conditions must be fulfilled for this process to happen:
    -> there must be contact between the fluorophore and the triplet quencher
    -> the rule of conservation of energy must be fulfilled, i.e. the energy difference between T1 and S0 of the fluorophore has to match that of the respective states in the quencher
  • a very efficient triplet quencher is oxygen having a stable triplet state
  • a fluorophore in the triplet state and triplet state oxygen can exchange an electron upon contact (orbital overlap), bringing both into the singlet state
  • triplet quenching by oxygen thus produces highly reactive singlet oxygen, which in turn may (photo-)oxidize (bleach) the dye
53
Q

Reversible photo chemistry

A
  • there is another dark state in the fluorescence of molecules: radical ionic states
  • these radical ionic states are typically a result of a two-photon absorption process: The first photon brings the fluorophore into an excited state, the second one ionizes the fluorophore from there
  • in order to shorten these dark states, a reducing/oxidizing system (ROXS) can be applied, being just a mixture of an oxidizing and a reducing agent

Strukturanalyse (11 decks)

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