lec 8- fluorescence spec Flashcards
Fluorescence spectroscopy relies on?
UV light is absorbed and re-emitted at a longer wavelength (fluorescence
in proteins which residue fluoresces
, tryptophan fluoresces
Commonly used in which 4 techniques?
3˚ and 4˚ structures
Measuring distances
Catalytic studies
Fluorescence microscopy
Excitation excites? and causes what?
Excites the electron in a chromophore into a higher energy state.
How do electrons return to ground state after excitation?
Transition vibrations
Rigid chromophores tend to have what type of transition vibrations?
Limited range, not possible to return to ground state by vibrations alone.
Instead they undergo: radiative transition (They lose energy by radiation, or light …. A portion of the absorbed energy is re-emitted )
Radiative transition?
They lose energy by radiation, or light …. A portion of the absorbed energy is re-emitted
Examples of natural fluorescent things?
Fluorescent minerals
Aequoria Victoria (GFP)
Fluorescent fish
Biophosphorescence
needs initial light source to activate, but then continues to “glow” after the light source is removed
Quinine?
Added to tonic water in order to prevent malaria.
example of a fluorescent compound
Quinine absorbs at?
460nm
Properties of fluorophores
- chromophore
- delocalised electrons - intense U.V. absorption bands
- ridged
- short excited state
Mechanism in which fluorescence is measured?
Quantum yield
where, Q= No. of photons emitted/” absorbed
Maximum q value?
1
Q is affected by:
Internal factors: distribution of vibrational levels
External factors: quenching
Still large q value?
0.1
Fluorophores are sensitive to?
environments»> quenched
most useful emission spectra?
Trp q= 0.13
fluorescent tags method of detecting proteins ??
The protein of interest is cloned into a vector, so that when it is expressed, it is attached to the fluorescent protein.
When cells are transfected with the DNA (a), when the protein of interest is expressed, so will the fluorescent protein and this can be detected by microscopy.
ethidium bromide is ??? how it works
tag ,
intercalates between the bases of DNA, and glows under UV lights – you can use a UV light box to see DNA bands on a gel
Ethidum bromide issue? therefore ?
carcinogen, often replaced with SYBR green
Fluorescin?
tag
Acridine orange works by?
Tag again
intercalates between bases of DNA – used as a cell-cycle marker
Spectrofluorimeter works by?
light source
passes through a monochromator (which splits the beam of light into different wavelengths)
through a sample (Light (uv) absorbed then emitted at a longer wavelength which is detected by a photomultiplier)
Why are two monochromators required in a Spectrofluorimeter
- To select wavelength of light required for excitation
2. To select for the wavelength of emitted light
At what angle is the emitted radiation detected at?WHY?
The emitted radiation is detected at 90 degrees to the direction of the incident light beam … this is to avoid inadvertent detection of the incident beam.
Cuvette most have what property in order to allow what?
the cuvette must have 4 clear sides to allow light through
Uses of fluorescent spectroscopy
Structural studies – tertiary and quaternary protein structure
Using tryptophan
FRET: Measuring distances within proteins and complexes
Binding / catalytic studies using a fluorescent substrate
Fluorescence microscopy
What qualities within the quaternary structure of proteins can be detected using Structural fluorescence spectroscopy
Folding / conformational state / monomer association / ligand binding
changes in structure can cause what to happen to tryptophan fluorescence?
changes in intrinsic tryptophan fluorescence (used as a reporter group)
When does tryptophan fluoress more?
it fluoresces more when it becomes buried as it is less quenched by water.
Less exposed to the solvent.
Shorter wavelength
Emission wavelength is dependent on?
the emission wavelength is dependent on the environment of trp
Tryptophan absorption and emission range
tryptophan has an abs max of 280nm, and emission peak from 300-350nm
Environmental factors effecting emission peak of tryptophan
Solvent polarity (exposure to aqueous phase)
Proximity of protonated groups such as Asp or Glu
Quenching by iodine, acrylamide and nearby disulphide groups
Quenching by nearby electron deficient groups like –NH3, -CO2H and protonated histidine residues
Unfolding kinetics can be used to?
Can be used to study the kinetics of folding / unfolding protein.
The most common methods in order to inflict unfolding of a protein
chaotropic agents= change in pH
As a protein unfolds?
Tryptophan fluorescent intensity and maximum emission wavelength changes
FRET stands for ?
fluorescent or Förster resonance energy transfer
FRET does what?
measures distances within proteins
FRET works by ?
Energy being transferred by resonance
Usually FRET works with?
engineered flours rather than trp
When can FRET occur?
can occur when emission spectrum of one fluorophore overlaps with the absorbance spectrum of a second fluorophore
Distance which is best appropriate for FRET?
occurs best when distance between fluorophores is 10-80A
The FRET efficiency depends on:
- The distance between the donor and acceptor
- The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum
- The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment
Uses of FRET?
- Spectroscopic ruler
- Can probe topology of membrane proteins
- Can study movement in contractile proteins
- Can determine changes upon receptor-ligand binding
Advantages of FRET?
quick and simple
Limitation of FRET?
Limited range (10-80Å) Need two fluorophores (trp often used as a donor, often the acceptor is engineered) Engineering a fluorophore that doesn’t alter the system that you’re studying is a challenge.
In ligand binding/catalytic studies what Is fluorescently labelled
Substate/ligand
When the substate/ ligand binds
change in fluorescent (either cause or take away)
Fluorometers work by?
The light source excites the fluorescent tag which is detected by detector
LIGAND BINDING KINETICS
CAN CALCULATE KD AND RECEPTOR AFFINITIES
How does fluorescence microscopy work?
The fluorescent tag (e.g. GFP / FITC) on the antibody is excited by the specified wavelength of light from the light source, which is directed to the sample
How Is fluorescence microscopy detected?
The emitted light travels to the eyepiece (and camera) which it is photographed.
Why are membranes difficult to study?
Expressing and purifying in membrane is tricky
Crystallising in membrane is difficult
Too large for NMR
Electron microscopy is improving
Chloroplast coupling factor catalyses?
synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate in the thylakoids of higher plants
