Lecture 4

Question 1

Which motor protein moves vesicles and other organelles towards the +end of the microtubules

Answer 1

Kinesin

Question 2

Motor protein transport of organelles doesn’t require ATP, T or F

Answer 2

F – it does

Question 3

Polyacrylamide gel electrophoresis and Western blotting can also be used to determine protein interactions, T or F

Answer 3

T

Question 4

Where in the kinesin structure does the organelle attach to

Answer 4

The other end of the long coiled-coil that holds the two motor heads together

Question 5

What attribute of protein interactions can be determined using competitive ELISA

Answer 5

How strongly an antibody binds to the protein of interest

Question 6

Explain how fluorescent resonance energy transfer can be used to investigate protein interactions

Answer 6

Recombinant fusion of protein X and Y to separate fluorescent proteins that absorb and emit certain wavelengths of light allows you to determine if X and Y interact/bind. By correlating the wavelength emitted by the fluorescent protein attached to X with the wavelength of light needed for fluorescence of protein Y you can activate protein Y fluorescence if it is in close proximity to X (i.e. it is bound). I.e. if shining light needed for fluorescence in protein X leads to the appearance of light that is given off as a result of protein Y fluorescent you can determine that X and Y interact

Question 7

What physical phenomenon of atoms/molecules drives the random movement of kinesin heads

Answer 7

Brownian motion

Question 8

What phenomenon is FRET said to rely on

Answer 8

Paired fluorescence

Question 9

Explain the structure of muscle myosin

Answer 9

Muscle myosin is a dimer with two identical motor heads which act independently. Each myosin head has a catalytic core and an attached lever arm. A coiled-coil rod ties the two heads together, and tethers the myosin heads to the thick myosin filament

Question 10

How does indirect ELISA differ from the direct approach

Answer 10

Indirect ELISA uses a secondary antibody that binds to the primary antibody to visualise interaction with the target protein. This secondary antibody is the one that contains the tag/label

Question 11

Explain how surface plasmon resonance can be used to investigate protein interactions

Answer 11

Light is shone onto a gold film with plasmon-induced evanescent electric filed which extends beyond the gold film by a distance that corresponds to the wavelength of the light shone onto it. Changes in the composition of the environment causes a measurable change in the resonance angle rpduced by the light refracting off the gold-coated prism. A solution containing proteins that might interact with the immobilized bait protein attached to the film is allowed to flow past the biosensor surface. Interacting proteins bind to the bait causing a change in the resonance angle of the light which is measured by a sensor

Question 12

How can you calculate the equilibrium constant for the interaction between two proteins using radioactive ligand binding data

Answer 12

K = [AB] / [A][B] = kon / koff

Question 13

How do kinesins transport organelles along the microtubules

Answer 13

Both kinesin heads initially contain tightly bound ADP, and move randomly. When one of the kinesin heads encounters a microtubule, it binds tightly. Microtubule binding causes ADP release from the attached head. ATP then rapidly enters the empty nucleotide binding site which triggers the neck linker to zipper onto the catalytic core. This action throws the second head forward, and brings it near the next binding site on the microtubule. The attached trailing head then hydrolyses the bound ATP, releasing a phosphate. As the neck linker unzippers from the trailing head, the leading head exchanges its nucleotide and zippers its neck linker onto the catalytic core, and the cycle repeats.

Question 14

What is meant by sandwich ELISA

Answer 14

The target protein antigen is captured by multiple antibodies. The primary antibody binds to the protein of interest but then another antibody that is also complimentary to the antigen, also binds to it. A secondary antibody is then introduced that binds to the primary antibody(s) and contains the tag/label

Question 15

What would be seen in the binding curve of a protein ligand that interacts more strongly with its target

Answer 15

Its binding curve will reach a maximum and plateau quicker as less ligand will be required for 50% binding saturation

Question 16

How can the yeast-2-hybrid screening technique be used to identify interacting proteins

Answer 16

Mix the prey and bait proteins produced by the yeast. The bait will bind to the reporter gene (HRP, GFP, ?Gal etc.) promoter via the fused binding domain. Prey fusion proteins containing protein(s) that interact with the protein of interest will bind to the bait via the protein of interest. Presence of both the BD and AD will lead to regulation of the reporter gene. The regulation of the reporter gene can then be used to identify the which yeast cells have the correct combination of interacting proteins. You can then isolate the plasmid from the yeast and sequence the plasmid to identify the gene the codes for the interacting protein

Question 17

Explain the enzyme-linked immunosorbent assay technique to determine protein interactions

Answer 17

The antigen, i.e. protein of interest is attached to a 96 well plate. Then a primary antibody is then added to the plate that binds to the protein of interest. In direct ELISA, this primary antibody carries a label that allows it to be visualised.

Question 18

The binding constant can be determined from SPR using association and dissociate rate constants, T or F

Answer 18

T

Question 19

What prevents the actin filament from sliding back after being released by the myosin motor head

Answer 19

There are many other myosin molecules also attached to it that hold it under tension

Question 20

Phage display is referred to as a genetic technique to investigate protein interactions, explain how it works

Answer 20

Random peptide sequences from a peptide library are integrated into a phage plasmid between restriction sites within the coat protein region. This means that the potential interacting peptides will be expressed in the coat of the virus allowing for potential interactions with the protein of interest. A solution containing the millions of potentially interacting peptides are added to the target protein. Unbound phages without interacting peptide can be removed using a buffer wash whilst interacting proteins will bind to the protein of interest. Addition of high concentration salt solution can the allow elution of bound phages that indicate interacting peptides. The phages that bind to the protein of interest can then be isolated, multiplied, DNA extracted and the sequence of the interacting peptides determined.

Question 21

What features can you derive from the binding curve of ligand concentration against binding

Answer 21

Max binding stoichiometry - how much of the interacting protein can bind to the protein of interest. Association constant – affinity of interacting protein to the protein of interest

Question 22

How do myosin and actin filaments interact

Answer 22

Myosin heads initially contain bound ADP and phosphate, and have weak affinity for actin. Once one of the heads docks properly onto an actin subunit, phosphate is released. Phosphate release strengthens the binding of the myosin head to actin, and also triggers the force-generating power stroke that moves the actin filament. ADP then dissociates, and ATP binds to the empty nucleotide binding site. This causes the myosin head to detach from the actin filament and ATP hydrolysis occurs. This re-cocks the lever arm back to its pre-stroke state. The coiled-coil arm thus stores the energy released by ATP hydrolysis, and the cycle can repeat.

Question 23

Which proteins are required for vesicular and organelle transport along the microtubules

Answer 23

Dynein and kinesin

Question 24

Surface plasmon resonance cannot be used to image protein interactions in real-time, T or F

Answer 24

F – it can, huge advantage

Question 25

Explain how the tools for a yeast-2-hybrid screen can be created

Answer 25

Separate the binding domain (BD) and activation domain (AD) of a regulatory protein that normally binds to the DNA to upregulate expression of a reporter gene. Fuse the protein of interest to the BD of the regulatory protein, now referred to as the bait. Fuse the AD of the regulatory protein with various peptide sequences from a cDNA library containing potential interacting peptides. These fusion proteins are referred to as the prey. Yeast cells are then cotransformed with BD and AD expression plasmids. Each transformant has the bait plasmid and one of the plasmids from the prey library.

Question 26

How can electron microscopy be used to directly visualise interacting proteins

Answer 26

EM uses a negative stain or vitreous ice (cyro-EM) to preserve the specimen. Image analysis is then employed to build up an average protein structure. EM allows visualisation of changes in the protein i.e. if it becomes thicker in certain parts when a binding partner is added. This allows you to determine where this potential binding partner is binding - which domain

Question 27

Which motor protein moves vesicles and other organelles towards the -end of the microtubules

Answer 27

Dynein

Question 28

Describe the structure of kinesin

Answer 28

The kinesin motor protein is a dimer that consists of two identical motor heads. Each head consists of a catalytic core and a neck linker

Question 29

Explain how to construct a radioactive binding curve to investigate protein interactions

Answer 29

Attach the protein of interest to beads by incubating the beads with an excess of the protein ligand. Unbound protein ligand can then be separated using centrifugation. Increasing the amount of ligand will increase the amount of ligand binding to the protein of interest leading to saturation of the binding sites. You can then record the amount of ligand bound per binding site and plot this against the concentration of ligand added.

Question 30

How does competitive ELISA differ from other enzyme-linked immunosorbent techniques

Answer 30

A labelled antibody complimentary to the target protein binds to the antigen. Then another antibody is introduced that competes for binding to the primary antibody. An enzymatic reaction between the antibodies gives a colour