Fluorescence

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What is S1V0*?

The energy level an electron has to fluoresce from to return to the ground state

How does the number of free electrons affect fluorescence?

The more free electrons, the more that are available to absorb a photon and be excited and the stronger the fluorescence

Why is FRET considered to be a spectroscopic ruler?

Because energy transfer is dependent on distance

Why do we no longer use ethidium bromide to identify/quantify DNA?

Because it is carcinogenic

Why is FRET considered to be a quenching process?

Because it stops the donor from fluorescing

How does size affect anisotropy?

Bigger molecule, slower rotation, increased anisotropy

How do non-polar/hydrophobic environments affect fluorescence? (4)

Blue-shifted, shorter wavelength, higher energy, increased quantum yield

How can protein kinase A activation be monitored?

By loss of FRET

What can Fura dyes be used to measure?

Ca2+ concentration

What is fluorescence sensitive to?

Environment

What is quantum yield sensitive to? (2)

Environment and quenching

What did FRET results show about protein kinase A activation when cAMP bound?

FRET decreased because subunits dissociated and less energy was transferred due to increased distance

What did FRET results show about clathrin structural changes?

FRET increased when clathrin monomers formed cage structure because the light chain contracted bringing fluorophores closer together

What does low anisotropy correspond to?

Fast rotation, less polarisation retained

What was added to each type of subunit in protein kinase A?

Fluorescein (donor) to catalytic subunits and rhodamine (acceptor) to regulatory subunits

Name the two main types of photoiluminescence

Fluorescence and phosphorescence

What does FRET stand for?

Fluorescence resonance energy transfer

What form is energy released as in dynamic quenching?

Heat

Why do you want minimal energy input to give a fluorescent signal?

Inputting lots of light can heat up your sample and denature it

What are the disadvantages of intrinsic biological fluorophores? (3)

Limited number, not very strong fluorescence relative to extrinsic fluorophores, may not be in the area you want to study

What is reacted with thiols to add a fluorophore?

Maleimide

What can ANS be used to study?

Membrane fluidity

What can heavy metals that fluoresce be used to study?

Metal binding

What can anisotropy be used to monitor?

Motion of molecules

What time scale does relaxation from S1V0* occur on?

Nanosecond

Define fluorescence

The emission of light when a molecule in a photochemically excited (first) singlet state returns to the ground state

What is FRET?

The transfer of energy between two fluorophores

What does maximum absorbance occur at?

The wavelength that matches the energy gap between the ground state and the excited state

Which amino acid groups can have fluorophores added to them?

Thiols and amines

Which amino acid is the most strongly fluorescent?

Tryptophan

Name the amino acids that fluoresce

Tryptophan, tyrosine and phenylalanine

What does FRET require?

Two complementary fluorophores close in space (<100 A)

Why type of transfer occurs in FRET?

Unidirectional transfer

When do Fura dyes fluoresce?

When bound to Ca2+

When does phosphorescence occur?

When the electron that has absorbed a photon undergoes inter-system crossing and enters a triplet state

What concentrations can fluorescence detect?

nM, pM, fM

What is fluorescein sensitive to?

pH

What can quenching be considered as in terms of mechanism?

Any mechanism that decreases or stops fluorescence

How is energy lost in non-radiative transitions? (2)

As kinetic energy or heat

What is photobleaching?

Permanent loss of fluorescence due to photo-induced chemical modification of a molecule

What is luminescence?

Emission of light by matter in the absence of heat

What is incandescence?

Emission of light by matter that has been heated

What is chemiluminescence?

Emission of light due to a chemical reaction

How does the emission spectrum vary depending on the wavelength of excitation?

Emits across the same range of wavelengths but at different intensities

How does the speed of rotation affect the polarisation of the light emitted?

If a molecule can rotate quickly, unpolarised light will be emitted

What time scale does a small molecule in solution rotate on?

10^-15 seconds

What time scale does absorption occur on?

10^-15 seconds

What time scale does fluorescence occur on?

10^-9 seconds

How many tyrosine and tryptophan does albumin have?

18 and 1

What is the value of R0?

30-70 A

What is R0?

50% transfer efficiency distance

Why is fluorescence used to measure reactions and not absorption?

Absorption occurs too quickly whereas fluorescence occurs more slowly and on the same time scale

What causes photoluminescence?

Absorption of a photon

What can dynamic quenching be used to probe?

Accessibility of fluorophores

What FRET pair was used to study clathrin?

Alexa 555 (donor) and alexa 647 (acceptor)

Why is phosphorescence considered to be a type of quenching?

Although it emits a photon, it prevents energy being released as fluorescence

What does cAMP do to protein kinase A?

Causes subunits to dissociate

What does dynamic quenching involve?

Collision (transient interaction) between a fluorophore and a quencher

What is quenching?

Competing processes that induce non-radiative relaxation of excited state electrons to the ground state

What does measuring distance using FRET allow you to monitor?

Conformational changes

What can SYBR Green I be used identify/quantify?

DNA

What does the fluorescent properties of acridine orange depend on? (2)

DNA/RNA and double-stranded or single-stranded

What effect does increasing the probability of phosphorescence have on fluorescence?

Decreases its likelihood

What does static quenching involve?

Direct chemical interaction between a fluorophore and a quencher

What can FRET be used to study?

Distance between two fluorophores

What does quantum yield measure?

Efficiency of fluorescence

Describe the mechanism of fluorescence

Electron absorbs a photon - excited - decays through non-radiative transitions - relaxation back to the ground state accompanied by the emission of light

What does the spectrum of tyrosine and tryptophan in albumin look like and why?

Looks like tryptophan spectrum because energy is transferred

What does the spectrum of a free mixture of tyrosine and tryptophan look like and why?

Looks like tyrosine spectrum because energy is not transferred

Why is fluorescence considered to be a safe technique? (2)

Non-invasive (protein does not have to be modified and can be studied in its natural state) and operator-safe (no radioactivity)

How is quantum yield calculated?

Photons emitted/photons absorbed

What are two major properties of fluorophores and why?

Pi systems (delocalised electrons) and rigidity (less ability to lose energy via non-radiative transitions)

How do polar/hydrophilic environments affect fluorescence? (4)

Red-shifted, longer wavelength, lower energy, decreased quantum yield

Name three properties of fluorescence

Sensitive, speedy and safe

How does the wavelength and energy of the excitation light compare to the emission light?

Shorter wavelength, higher energy

How has FRET been used to study clathrin?

Site-directed mutagenesis in light chain introduces cysteines with chemically bound fluorophores

What does high anisotropy correspond to?

Slow rotation, more polarisation retained

How does the time scale of phosphorescence compare to the time scale of fluorescence?

Slower

Name two types of quenching

Static and dynamic

What is reacted with amines to add a fluorophore?

Sulfo-NHS esters

Give two examples of heavy metals that fluoresce and what they are analogues of

Terbium (Ca2+) and europium (Mg2+)

What does describing two fluorophores as complementary mean?

That the wavelengths of donor emission and acceptor excitation overlap

What is meant by fluorescent lifetime?

The average time a molecule stays in the S1V0* level before emitting a photon

What is anisotropy?

The degree to which the polarisation of light is retained after fluorescence


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