PROTEIN INTERACTIONS

Question 1

STRUCTURES OF INTERACTION PROTEINS - leucine zipper, zinc finger, B sheet, BHLH

IMPORTANT PROPERTIES

Answer 1

BHLH - helix that has been broken and loops back - forms 2 alpha helixes wrapped round the DNA.
Leucine zipper - 2 alpha helices wrapped round the DNA
Zinc fingers - 2 b strands with an A-helix
B Sheet - b strand bound to main strand of DNA

+ve so can interact with -ve protein

Question 2

Leucine zipper domain - purpose?

Answer 2

Dimers form a short coiled-coil sequence like a clothes peg on a washing line (the washing line being the DNA main sequence). Specific DNA recognition helix.

Question 3

Zinc finger domains - structure? What is it used for?

Metal ions in DNA binding? metal ion valencies?

Answer 3

Helps in stability of DNA, made up of tetrahedrally coordinated cysteine and histidine residues.
Metal ions are used commonly in binding of proteins - ions with different valencies bound by different numbers of amino acids

Question 4

AFFINITY CHROMATOGRAPHY. Method used to purify DNA binding proteins

Answer 4

Specific DNA added to a silica bead. All proteins are added. Unbound proteins will wash out. Salt added to separate bound sample from the beads.
Sample purified.

Question 5

DNA FOOTPRINTING - confirms a protein interaction sequence. Method?

Answer 5

A radioactive end label is added to a protein. DNA binding proteins are added. DNA is digested by a cleavage agent (DNAse) at random locations. The bound proteins will shield the DNA from being cut at particular places. Can compare bands by gel electrophoresis and the bands that are not present will have a DNA binding protein at that particular place, showing that the protein interacts with that specific DNA sequence.

Question 6

OTHER PROTEIN INTERACTIONS - SH2 domains, EF hand, SH3 domain, PH domain???

Answer 6

SH2 domain - binds to phosphorylated proteins
EF hand - binds to ca2+ / mg2+ for structural signalling
SH3 domain - binds to proline rich motifs and peptides
pH domain - binds to phospholipids

Question 7

SH2 DOMAIN - where do they originate from?

Answer 7

originate from Src (a protein tyrosine kinase), and bind to proteins by ionic interactions between the Hydrogen bonds of the SH2
(the phosphate is +ve and the a-acids in the SH2 domain protein are -ve)

Question 8

SH3 DOMAIN - how does it work? overall mechanism?

Answer 8

Aromatic residues that use a HYDROPHOBIC STACKING MECHANISM to bind the ligand.
bind 2 PROLINE GROUPS
Link together signalling complexes and have a structural role.

Question 9

pH DOMAIN - function? what do they do? how do they work?

Answer 9

Anchor proteins to the membrane to help with signalling. Found at the charged head group of phospholipids to anchor proteins to membrane.
Work on a HYDROPHOBIC/CHARGE interaction and allow membrane surface association.

Question 10

TAGGING!! Why is this done? METHOD

Answer 10

Can tag protein of interest (protein X) in a solution with a sequence and then bind an antibody to protein X. Can then add protein X (bound by the antibody) to a column with beads coated in an antigen for the antibody. This eludes the antibody-bound protein X from the column and allows purification for further study.

Question 11

COMMON TAGS

Answer 11

For affinity chromatography - GST and Hexahistidine.

For immune precipitation - Flag / HA / Myc peptide.

Question 12

GST tagging - method of affinity chromatography

Answer 12

Protein of interest is fused to GST and cell extract added to column with glutamine-coated beads.
Elusion from the column using GLUTATHIONINE
Then SDSpage and western blotting

Question 13

MEASURING STRENGTH OF PROTEIN INTERACTIONS - methods.

Answer 13

Can use surface plasmon resonance, ELISA and live imaging

Question 14

YEAST 2 SCREEN HYBRIDISATION
use of a reporter gene
method
what are the bait and prey constructs?

Answer 14

an interaction between 2 proteins will result in transcription of a reporter gene. This allows survival on. a restricted media.
BAIT PROTEIN - the target protein and a DNA binding domain
PREY PROTEIN - the transcriptional activator and part of a large library of plasmids.
Once the protein interaction has been made and the surviving colonies have grown on the media, the plasmids in surviving colonies are sequences and the interacting protein is found.

Question 15

ELISA
enzyme linked immunosorbant assay
what is it for? method?

Answer 15

An interactive enzyme is used which binds the protein of interest.
An enzyme bound to an antibody is added that produces a signal e.g. fluorescent light when activated
The signal is produced once the enzyme has bound to the protein of interest and the substrate to the enzyme added - stronger interaction = more of signal produced

Question 16

FLOURESCENCE RESONANCE ELECTRON TRANSFER (FRET)

use of light of one colour to stimulate another protein

Answer 16

Protein X links a blue fluorescent protein and is stimulated with violet light to produce blue light.
Protein Y links to GFP and is stimulated by blue light to produce green light.

SHINE VIOLET LIGHT AND MEASURE GREEN LIGHT READINGS

measure protein interactions

Question 17

RADIOACTIVE BASED BINDING APPROACH

binding curve produced

Answer 17

a stronger interaction between the protein of interest and a radio active protein will produce a steeper radioactive binding curve.

Beads will be soaked in the protein of interest and the radioactive binding protein added - can wash and count how many bound materials there are on the beads

Binding curve represents association constant

Question 18

SURFACE PLASMON RESONANCE

Answer 18

Used to detect interactions between proteins by the change in resonance angle of light - this can be quantifyably measured by a detector

Can produce an interaction curve

Question 19

MEASURING INTERACTIONS
associattion rate

dissociation rate

equilibrium equation?

Answer 19

A + B = AB
association rate = association rate constant x [A] x [B]
= Kon[A][B]

AB –> A + B
dissociation rate = dissoc rate constant x [AB]
= Koff[AB]

@ equilibrium,
Koff [AB] = Kon [A][B]

Question 20

Visualising protein movements?
Kinesin and microtubules?
Microscopy

Answer 20

Can visualise the movements of kinesis by purifying it and attaching it to MICROTUBULES on a glass plate

LIGHT MICROSCOPY can be used - shows vesicle movement along microtubules. This required ATP and shows how it interacts with microtubules.