lecture 8

Question 1

What is Fluorescence and how does it occur?

Answer 1

• 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.

Question 2

What is an example of natural compounds that emit fluorescence ?

Answer 2

Aequoria victoria, biofluorescent jellyfish known for GFP

Question 3

What is Biophosphorescence?

Answer 3

needs initial light source to activate, but then continues to “glow” after the light source is removed

Question 4

what is Bioluminescence?

Answer 4

no need for an activating light source, chemicals release light on their own.

Question 5

How was gin and tonic discovered?

Answer 5

• Quinine added to tonic water to fight against malaria

- This anti- malarial tonic led to British colonials mix it with gin

Question 6

What are the 5 properties of fluorophores?

Answer 6

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)

Question 7

What is the equation used to measure fluorescence?

Answer 7

Q = Number of photons emitted / Number of photons absorbed

Question 8

What is the maximum fluorescence value?

Answer 8

1

Question 9

How is Q affected?

Answer 9

Internal factors: distribution of vibrational levels

External factors: quenching

Question 10

Do proteins fluoresce as we know that aromatic amino acids contain chromophores?

Answer 10

• low Q VALUES
• quenching - emitted light will be quenched by Trp
  Trp has the most useful emission spectra

Question 11

What is the exception to the protein fluorescence?

Answer 11

Tryptophan is the exception with a more useful emission spectra

Question 12

How can we label proteins?

Answer 12

fluorescent tags to enable detection

Question 13

What are the examples of fluorescent tags?

Answer 13

• GFP
• Ethidium bromide
• Fluorescin
• ANS - ANS
• Acridine orange

Question 14

How does a spectrofluorimeter work?

Answer 14

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.

Question 15

How can fluorescent spectroscopy be used?What are the 4 ways?

Answer 15

• Structural studies using Tryptophan
• FRET - measuring distance withe proteins and complexes
• Binding/ catalytic studies using a fluorescent substrate
• Fluorescence microscopy

Question 16

How does structural fluorescent spectroscopy used?

Answer 16

• Quaternary structure

- Changes in these structures can cause changes in intrinsic tryptophan fluorescence (used as a reporter group)

Question 17

How is the Trp fluorescence stopped in structural fluorescence spectroscopy?

Answer 17

Quenched with water

Question 18

How does ligand binging work?

Answer 18

• 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

Question 19

What is Fluorescence microscopy used for?

Answer 19

Commonly used laboratory technique to visualise specific proteins

Question 20

How does Fluorescence microscopy work?

Answer 20

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.

Question 21

Why are membrane proteins difficult to study in the thylakoid membrane of chloroplasts?

Answer 21

• Expressing and purifying in membrane is tricky
• Crystallising in membrane is difficult
• Too large for NMR
• Electron microscopy is improving

Question 22

What is the name of the technique used to study the chloroplast coupling factor?

Answer 22

FRET

Question 23

How does FRET work in understanding the chloroplast coupling factor?

Answer 23

• 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

Question 24

What is Rhodopsin?

Answer 24

light absorber in the discs of rod cells in eye.

Question 25

What does electron microscopy indicate about Rhosopsin?

Answer 25

Electron microscopy indicates Rhodopsin is a dimer in the membrane

Question 26

What are the two components of rhodopsin?

Answer 26

Light sensitive membrane receptor (opsin)

Covalently bound co-factor (retinal)

Question 27

How was rhodopsin analysed?

Answer 27

By Fret

Question 28

How was Rhodopsin analysed?

Answer 28

Rhodopsin monomers were labelled with florescent probes

Question 29

What is the detailed mechanism of how rhodopsin was analysed?

Answer 29

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.

Question 30

How is Fret used to determine Rhodopsin dimers/ oligomers?

Answer 30

Opsin transiently expressed in COS1 cells with yellow / cyan fluorescent protein attached.
CFP = donor
YFP = acceptor

Question 31

What is the 5HT3 / serotonin?

Answer 31

5HT3 / serotonin receptor

Question 32

What is the 5HT3 receptor ?

Answer 32

Ligand gated ion channel

Target for some antidepressants.

Question 33

How can ligand binding be determined for the 5HT3 receptor?

Answer 33

Fluorescein-GR-H fluorescent ligand (Gr-flu) 5-HT mimic was synthesised

Question 34

What happens with the synthesise ligand?

Answer 34

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.