lecture 8 Flashcards
What is Fluorescence and how does it occur?
- The excitation energy (usually UV or visible light) excites the electron in a chromophore into a higher energy state.
- Some rigid, inflexible chromophores, have a limited range of vibrational energy levels – and it is not possible for them to return to the 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 as light.
What is an example of natural compounds that emit fluorescence ?
Aequoria victoria, biofluorescent jellyfish known for GFP
What is Biophosphorescence?
needs initial light source to activate, but then continues to “glow” after the light source is removed
what is Bioluminescence?
no need for an activating light source, chemicals release light on their own.
How was gin and tonic discovered?
- Quinine added to tonic water to fight against malaria
- This anti- malarial tonic led to British colonials mix it with gin
What are the 5 properties of fluorophores?
Must be a chromophore:
Delocalised electrons (alternate single & double bonds, aromatic rings).
Intense U.V absorption bands (e.g. π to π* transition)
Rigid structure
Short excited state lifetimes (<10 –9 sec)
What is the equation used to measure fluorescence?
Q = Number of photons emitted / Number of photons absorbed
What is the maximum fluorescence value?
1
How is Q affected?
Internal factors: distribution of vibrational levels
External factors: quenching
Do proteins fluoresce as we know that aromatic amino acids contain chromophores?
- low Q VALUES
- quenching - emitted light will be quenched by Trp
Trp has the most useful emission spectra
What is the exception to the protein fluorescence?
Tryptophan is the exception with a more useful emission spectra
How can we label proteins?
fluorescent tags to enable detection
What are the examples of fluorescent tags?
- GFP
- Ethidium bromide
- Fluorescin
- ANS - ANS
- Acridine orange
How does a spectrofluorimeter work?
Emitted radiation (of longer wavelength) is detected by a photomultiplier tube Notice that there are two monochromators in this system: 1. To select wavelength of light required for excitation 2. To select for the wavelength of emitted light 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.
How can fluorescent spectroscopy be used?What are the 4 ways?
- Structural studies using Tryptophan
- FRET - measuring distance withe proteins and complexes
- Binding/ catalytic studies using a fluorescent substrate
- Fluorescence microscopy
How does structural fluorescent spectroscopy used?
- Quaternary structure
- Changes in these structures can cause changes in intrinsic tryptophan fluorescence (used as a reporter group)
How is the Trp fluorescence stopped in structural fluorescence spectroscopy?
Quenched with water
How does ligand binging work?
- The ligand or substrate is fluorescently labelled
- When receptor-ligand binding or enzyme-substrate binding occurs, there is a change in fluorescence.
- This is detected with a spectrofluorimeter
What is Fluorescence microscopy used for?
Commonly used laboratory technique to visualise specific proteins
How does Fluorescence microscopy work?
A labelled (fluorophore or colour) secondary antibody binds to the primary antibody, and allows visualisation by microscopy
- 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
The emitted light travels to the eyepiece (and camera) which it is photographed.
Why are membrane proteins difficult to study in the thylakoid membrane of chloroplasts?
- Expressing and purifying in membrane is tricky
- Crystallising in membrane is difficult
- Too large for NMR
- Electron microscopy is improving
What is the name of the technique used to study the chloroplast coupling factor?
FRET
How does FRET work in understanding the chloroplast coupling factor?
- Fluorescent probes were placed in different parts of the molecule and 30 inter-probe distances measured (FRET)
- Allowed understanding of quaternary structure: relative positions of subunits and their orientation with respect to the membrane
What is Rhodopsin?
light absorber in the discs of rod cells in eye.
What does electron microscopy indicate about Rhosopsin?
Electron microscopy indicates Rhodopsin is a dimer in the membrane
What are the two components of rhodopsin?
Light sensitive membrane receptor (opsin)
Covalently bound co-factor (retinal)
How was rhodopsin analysed?
By Fret
How was Rhodopsin analysed?
Rhodopsin monomers were labelled with florescent probes
What is the detailed mechanism of how rhodopsin was analysed?
When they are as monomers … the florescence from the donor cannot “reach” the acceptor in the other monomer.
When they bind and become a dimer, they are close enough for the resonance energy to transfer to the acceptor fluor and we get a change in the wavelength released.
How is Fret used to determine Rhodopsin dimers/ oligomers?
Opsin transiently expressed in COS1 cells with yellow / cyan fluorescent protein attached.
CFP = donor
YFP = acceptor
What is the 5HT3 / serotonin?
5HT3 / serotonin receptor
What is the 5HT3 receptor ?
Ligand gated ion channel
Target for some antidepressants.
How can ligand binding be determined for the 5HT3 receptor?
Fluorescein-GR-H fluorescent ligand (Gr-flu) 5-HT mimic was synthesised
What happens with the synthesise ligand?
This fluorescently labelled ligand emits fluorescent light at 520nm when it is not bound to the 5HT receptor
However, when it does bind to the 5HT receptor, the fluorescent signal at 520nm is blocked and the amount of emitted light is decreased as you can see in the graph.
