Lecture 16 - Protein Interactions II

Question 1

colocalisation:

Answer 1

idea of if two things are occupying the same space, they are probably interacting but this isn’t always necessarily true

using colocalisaiton of fluorescent signal from different antibodies for the proteins of interest does not prove interaction - just that they are in the same physical space

Question 2

what is proximity ligation assay (PLA)?

Answer 2

PLA is an imaging-based method that only produces a signal if the proteins are close enough together

like IP it uses antibodies that recognise proteins we are interested in

important to use antibodies of different species, secondary antibodies play a role in PLA as instead of fluorophores, we have DNA oligonucleotide sequences attached to the end

Question 3

how does PLA function?

Answer 3

if the proteins are close enough together, the secondary antibodies will be close enough together

if the secondary antibodies are within 40nm of each other, connector oligos will be able to bind between the oligos attached to secondary antibodies

these oligos are able to be lighted and joined together like plasmids in cloning

this forms a circular DNA strucutre

this DNA is then transcribed as it is as a circle, it keeps going round and round to make a larger transcript (this is called rolling amplification)

with long repetitive oligo sequences being generated, oligonucleotide sequences with fluorescent molecules hybridise and bind

this can then be seen in a confocal microscope to detect the signal, every signal is the conformation of an interaction between two proteins within the cell

Question 4

oligos will ligate if…

Answer 4

… the secondary antibodies they are attached to are close enough together (40nm)

Question 5

what is important to consider when using PLA?

Answer 5

important to include controls to check that the signal is specific since it appears as dots
- this includes running the primary antibodies by themselves and then no primary antibodies used
- there is always a level of background staining where the secondary antibodies will just bind to things in the cell

• important also to consider what is a positive signal - are we looking for large discrete dots or smaller dots

Question 6

FUNCAT-PLA:

Answer 6

PLA approach can be modified to look at newly synthesise proteins and their localisation - this is called fluorescence non-canonical amino acid tagging with proximity ligation assays (FUNCAT-PLA)

• requires a slight modification where they add a molecular tag to the methionine amino acid: with this incorporated, a combination of an antibody against the protein of interest and the tag allows for the localisation of newly synthesised proteins - can also be used for protein modifications

Question 7

PLA allows for:

Answer 7

interactions between native proteins to be shown through proximity and allows doe localisation of these interactions within cells

Question 8

PLA can be used in a variety of situations:

Answer 8

protein interactions, new protein synthesis & protein modifications

Question 9

FRET:

Answer 9

a technique that allows for interrogation of very close interactions of less than 10nm

Question 10

how FRET works:

Answer 10

FRET is where the energy generated from one fluorescent protein is used to excite another fluorescent protein , this causes a change in the colour of the fluorescent signal

requires fluorescent proteins to be close together so it shows they must be interacting

Question 11

when does fluorescence occur?

Answer 11

fluorescence occurs when light of particular wavelengths excite the molecule - this is called the excitation spectra, there is normally a peak wavelength that excites the molecule the most

Question 12

how do we normally excite the flurophore and what does it cause?

Answer 12

using a laser which causes light to be given off from the molecule across a range of wavelengths - this is called emission spectra [the emission is what we pick up on in the microscope]

Question 13

____ uses the properties of fluorescence by pairing the emission of one molecule with the excitation of another

Answer 13

FRET

Question 14

why, in FRET, does one fluorescent molecule excite another?

Answer 14

this is because there is a spectral overlap that exists between the two fluorophores between the emission of the donor molecule and the excitation of the acceptor

Question 15

in order for FRET to work:

Answer 15

there needs to be fluorescent proteins attached to the proteins of interest

Question 16

what is the most common pair of fluorophores and why are they the most common?

Answer 16

CFP & YFP, this is because they have the correct overlapping spectra

Question 17

FRET can be used in multiple situations:

Answer 17

• can be used to measure the distance between proteins when they react (fluorescence strength is dependant on distance)
• can be used for changes in protein conformation and for determining cleavage sites or sequences for enzymes

Question 18

considerations when using FRET:

Answer 18

• the fluorescent tags need to be appended to the end of the proteins [if the interaction site is distant form the ends, this may mean that the tags are too distant to allow for FRET]
• the fluorescent tags can also be large and affect the conformation of the protein and its interactions: may get in the way of the interaction making it less stable

Question 19

what may be a good to undertake before FRET?

Answer 19

may be good to undertake other interaction assays before using FRET

Question 20

FRET summary sentence:

Answer 20

FRET is a technique that allows tight interrogation of protein interacts using energy transfer between fluorescent molecules and detection of a signal