Protein-Protein Interactions Notes

Protein-Protein Interactions
The first question is why would we want to know about protein-protein

interactions? This kind of information would allow us to determine if a protein is in a
complex, a metabolic of signaling pathway, or involved with other structural proteins.
Sometimes a protein is identified by one function and then is found to be part of a
complex with a structural or unknown role. Many of the complexes are stable such as the
electron transport complexes, protein translocation complexes, nuclear pores etc., and
some are transient such as the binding of a docking protein to ribosomes translating a
secreted protein to take is to the ER or a G-protein involved in signaling.
There are three methods “commonly” used to look for protein-protein interactions,
Yeast two hybrid, pull down or co-purification and fluorescence resonance energy
transfer. We will discuss each of these.
Yeast Two Hybrid

The yeast two hybrid system is based on the presence of two domains in a
transcription activator, an activation domain and a binding domain. The process is we are
going to clone the protein of interest with one of the domains attached to it. This would
be called the bait protein. We then take the genome of the organism and clone the other
domain to all of them. This represents the prey proteins. These are then cloned into
separate yeast. The yeast are allowed to mate and transfer plasmids. Selection can be
made by using auxotrophs and providing genes that make amino acids. This allows the
selection of those cells that contain both plasmids. Once we have the cells with both
plasmids the cells that have interactions between the proteins will express the reporter
enzyme and they can be detected. One example used here is the Lac Z gene. This is used
in blue white screening of the cells.
There are also systems for cytosolic and membrane proteins. In the membrane
system a split ubiquitin process is used. In this case ubiquitin can be split into two halves
with the c-terminal ubiquitin (Cub) and the N terminal ubituitin (NubG). In this case the
bait consists of the Cub protion linked to the protein of interest and followed by a
transcription factor. The prey proteins will have the NubG sections attached to them. It
is assumed that both of these are either membrane bound proteins or at least the bait is a
membrane bound proteins. Once these two proteins are expressed and interact through
the ubiquitin system the transcription factor is released by proteolysis by ubiquitin
binding proteins. This allows the transcription factor to enter the nucleus and the
reported protein to be expressed. A similar approach can be used for cytosolic proteins as
well. It would seem to me that as long as the transcription factor can be released this
would also work for some portions of organelles.
Doing this on a genomic scale takes a little more effort. The genome is basically
cloned with both the bait and prey molecules and put into mating strains of yeast. When
they are mixed together the strains mate and mix the plasmids. The diploid cells are then
selected for those that survive some screening process. The plasmids in the survivors can
then be sequenced to determine which proteins are interacting.
Problems
There are several problems with this system. The first is that many false positives
and false negatives are found. Another problem deals with the proteins that are being
screened. They may not express well in the yeast system or they may not interact
properly when linked to the two domains. There is also the possibility that the
interactions are too transient to be measured. And lastly there are difficulties with
multiple subunit complexes since two of the subunits may not be close enough for the
two domains to come together.
Co-purification and Immuno-trapping.

This method is based on the concept that if the proteins are interacting in the cell
they may stay together long enough to be purified together. The assumption is the the
interaction will be long enough and strong enough to identify. The process is to tag one
protein so that it can be pulled down with beads or columns containing antibodies to the
tag. The protein is expressed in the cell, the cells lysed, and the bait protein purified by
magnetic beads or on a column. Once the other proteins are washed off the protein of
interest and its friends are removed by denaturation and analysis by LC/MS/MS. This
slide is just to remind you of the many tags that are available for this process.
Fluorescence Resonance Energy Transfer (FRET)

In this method we are going to use the ability of one molecule to transfer its
energy to another by emission of light and the uptake of that light by the other molecule.
The emission of light absorbed is called fluorescence. When a second molecule whose
absorption spectrum overlaps with the emission of the first molecule there can be a
transfer of the light energy to the second molecule. If the second molecule is fluorescent
then the light will be emitted by the second fluorescent molecule. The other possibility is
that the second molecule only absorbs the energy and does not emit it resulting in
quenching.
This process is highly distance dependent following a r-6 distance dependence.

The efficiency of transfer can be determined from the quantum yield of fluorescence with
and without the acceptor. This can be related to the distance by the formulas on the slides.
In this system the proteins of interest are tagged with one or the other molecule. They are
introduced into the system and the cells are illuminated with the excitation wavelength of
the first molecule and fluorescence is look for from the second molecule. If it is observed
then they are close together. The problem is how do we label them with fluorescent
molecules? If there is a way to look at the interactions in vivo we can chemically label
the proteins and observe the interaction. A more interesting process is to use various
forms of the green fluorescent protein and clone the proteins with one or the other
attached. We then look for the energy transfer in vivo through a microscope.
Problems
As usual there are problems. We have the usual problems of protein expression.
If an antibody based method is used for pull downs we have the general problems with
antibodies. Lastly fusion proteins may not work due to stearic hindrance or improper
folding.

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Protein-Protein Interactions

The first question is why would we want to know about protein-protein

Yeast Two Hybrid

Co-purification and Immuno-trapping.

Fluorescence Resonance Energy Transfer (FRET)

This process is highly distance dependent following a r-6 distance dependence.

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