Fluorescence
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