Lecture 3

Question 1

What post-translational modification targets proteins for transport to the nucleus of the cell

Answer 1

SUMOylation

Question 2

What does SDS-PAGE stand for

Answer 2

Sodium Diethyl Sulphate Poly-Acrylamide Gel Electrophoresis

Question 3

The poly-acrylamide gel has a sieving effect and protein migration through the gel in the presence of SDS is proportional to molecular mass, T or F

Answer 3

T

Question 4

Which two amino acids can be phosphorylated

Answer 4

Serine and threonine

Question 5

Describe how 2D gel electrophoresis can be used to separate proteins

Answer 5

A stable pH gradient is created in a commercially available gel. Proteins are introduced to the gel and run along it until they reach the pH that corresponds to their isoelectric point. At this pH the proteins become uncharged and no longer run along the gel.

Question 6

How can DNA footprinting be used to confirm DNA binding proteins

Answer 6

Firstly, a DNA sequence is radioactively labelled. This DNA sequence is then subjected to hydrolysis which produces all possible DNA sequence lengths with an ever diminishing molecular weight. However, some size fragments will be excluded from the fragment profile due to masking by the bound radiolabel. These specific fragments are unique to each DNA sequence and will be represented by a gap in the DNA binding profile. This acts as a fingerprint and allows the identification of the sequence.

Question 7

Explain the main differences between equilibrium and velocity density centrifugation

Answer 7

Velocity based density centrifugation involves the extraction of the different density fractions based on how long it takes them to deposit at the bottom of the tube whereas equilibrium based density centrifugation relies on prolonged high speed centrifugation to separate the different density organelles within the steep sucrose gradient.

Question 8

By what bonding do side chains of the helices of leucine zippers directly contact the DNA bases

Answer 8

Via hydrogen bonding

Question 9

Describe the process of affinity chromatography

Answer 9

Cytosol is added to a column containing beads with a covalently attached substrate. Enzymes for the substrate that is bound to the bead will bind irreversibly if the substrate is non-hydrolysable. Other proteins in the cytosol will pass straight through the column and only the enzyme will be retained. The bound protein(s) can then be eluted from the column using a competing ligand to dislodge the affinity interaction. This allows separation of specific substrate binding proteins from the cytosol

Question 10

Outline the typical methods used in protein purification

Answer 10

Intitially you start with ion exchange chromatography which acts as a rough separation method based on charge. The fractions produced from ion exchange chromatography are then used in gel filtration chromatography to separate the fractions further, based on size. In the final stage, these fractions are then separated further using affinity chromatography. A combination of two or three different chromatography techniques is used to separate pure proteins.

Question 11

What defines the working range of the resin in gel filtration (size exclusion) chromatography

Answer 11

The sieving effect of the beads due to the pore size. Proteins too large are excluded from moving through the column

Question 12

What phenomena is affinity chromatography said to rely on

Answer 12

Tight interactions (enzyme-substrate binding)

Question 13

How is the starting cell homogenate obtained in differential centrifugation

Answer 13

Blending, sonication or grinding with a pestle and mortar

Question 14

What change in molecular weight is seen as a result of phosphorylation

Answer 14

+95Da

Question 15

What is coupled mass spectrometry

Answer 15

Coupled mass spectrometry involves subjecting the peptide fragments to further digestion and then fragmentation using an electric field. The mass spectrometer can than analyse and determine the amino acid composition of the peptides.

Question 16

What is the role of SDS in the separation of proteins in SDS-PAGE

Answer 16

Sodium Diethyl Sulphate is a negatively charged molecule that repels proteins and causes them to straighten out and thus allows the proteins to move easily through the polyacrylamide gel

Question 17

Other than determining the identity of unknown proteins, what other use is there for mass spectrometry

Answer 17

Analysing post-translation modifications such as serine/threonine phosphorylation. This causes a change in molecular weight which can be picked up by the mass spec

Question 18

What two effects can ubiquitination have and how are these different pathways encoded

Answer 18

Poly-ubiquitination marks proteins for degradation whereas mono-ubiquitination directs protein recycling

Question 19

Explain how affinity chromatography is used to investigate interacting proteins and how it is achieved using pulldown proteins

Answer 19

Recombinant technology is used to create a fusion protein consisting of the protein of interest and glutathione-S-transferase (known as a pulldown protein). The fusion protein and cytosol is then added to a column containing glutathione coated beads. The GST fusion protein will bind to the glutathione coated beads together with any proteins in the cytosol that interact with the protein of interest. Once the cytosol has passed through the column and interacting proteins have bound together with the protein of interest to glutathione beads, a solution of free glutathione is added to the column to elute all of the bound proteins. These can then be separated using SDS-PAGE and identified using mass spectrometry.

Question 20

Describe the process of ion exchange chromatography

Answer 20

Diethylaminoethyl (DEAE) beads are added to a column in addition to negatively charged proteins. The proteins will bind to the positively charged DEAE beads. An increasing concentration of salt is then added to the column to displace the proteins form the beads. Less negatively charged proteins are displaced and release from the column first, at lower salt concentrations. More negatively charged proteins will be displaced later at higher salt concentrations. This allows you to separate fractions based on charge.

Question 21

Why is it often seen the 50kDa proteins are found in the middle of an SDS-PAGE

Answer 21

50kDa corresponds to the average proteins molecular weight (due to 500 residues) and so with most proteins being found in and around this weight it is a reasonable middle of the SDS-PAGE

Question 22

Explain how mass spectrometry can be used to determine the identity of unknown proteins

Answer 22

The isolated protein is incubated with proteases to digest it into peptides. These small peptides are then allowed to run in a mass spectrometer which ionises them. The mass spec machine then compares the recordings from the peptide fragments with a database of all known peptide combinations in order to determine the identity.

Question 23

Peroxisomes, lysosomes and mitochondria are contained in the second pellet during differential centrifugation, T or F

Answer 23

T

Question 24

What is contained in the pellet after the first centrifugation step in differential centrifugation

Answer 24

Whole cells, nuclei and cytoskeleton

Question 25

Give an example of a protein that is post-translationally modified by hydroxylation and which amino acid is hydroxylated

Answer 25

Proline residues in collagen are hydroxylated

Question 26

Other than GST, what other tag can be used in affinity chromatography to investigate protein interactions

Answer 26

Hexa-histidine (6xHis)

Question 27

What is the purpose of boiling protein samples with mercaptoethanol in the first stage of SDS-PAGE

Answer 27

Boiling with mercaptoethanol breaks the disulphide bonds between cysteine residues

Question 28

Explain the process of velocity sedimentation type density centrifugation

Answer 28

A test tube containing a stabilising gradual sucrose gradient is established whereby highest sucrose concentrations are found at the bottom of the tube. Addition and centrifugation of the cytosol separates the organelles based on density. Heavier organelles will sediment quicker and lighter ones will take longer. Organelles deposit at the sucrose concentration that equates to their own density. A hole is punctured in the bottom of the test tube and organelles can be collected from the bottom in various fractions depending on their density.

Question 29

Describe the basic structure of a zinc finger domain and how it interacts with DNA

Answer 29

Zinc finger domains consist of an ? helix and 2 ? strands. They interact with the major groove of DNA and via guanylyl bases

Question 30

What is the enzyme responsible for the ubiquitination of lysine residues

Answer 30

Ubiquitin ligase

Question 31

Where is protein methylation often seen and which enzymes and amino acids are involved

Answer 31

Methylation of lysine and arginine residues is often seen in histones and are involved in epigenetics. Methyltransferase enzymes are responsible for this process

Question 32

Describe the quaternary structure of the leucine zipper domain

Answer 32

Leucine zippers contain two intertwined ? helices

Question 33

Describe the protein-fraction graph produced in affinity chromatography

Answer 33

Upon addition of the cytosol to the column there is an initial large peak in the relative protein amount which decays away as fraction numb increases. Addition of an eluting substance then causes another peak in relative protein amount due to elution of the bound protein from its substrate

Question 34

Other than epigenetics and in histones, what other role can acetylation play

Answer 34

Acetylation of tubulin stabilises the microtubules

Question 35

Coupled mass spectrometry is usually carried out after affinity chromatography, T or F

Answer 35

F – it’s usually carried out after 2D gel electrophoresis

Question 36

Palmitoylation and farnesylation are examples of lipidation, T or F

Answer 36

T

Question 37

Where/when is 2D gel electrophoresis commonly used

Answer 37

Proteomics and in complex samples

Question 38

Explain how tags are used in immunoprecipitation to identify interacting proteins

Answer 38

HA, Myc or Flag peptides are fused to the protein of interest. The cytosol containing potential interacting partners and the fusion protein is added to a column containing beads covered in a bacterial protein that binds strongly to antibodies. In addition, anti-HA, anti-Myc or anti-Flag antibodies are also added to the column. These antibodies will bind to the target protein, together with any interacting proteins and then will bind via their Fc domain to the bacterial protein on the beads. Once the protein of interest and interacting proteins are bound via antibody interactions to the beads they can be analysed by Western Blotting or Mass Spectrometry

Question 39

Explain how SDS-PAGE separates the different proteins

Answer 39

Once loaded into wells, the negatively charged proteins move towards to positive electrode. Smaller proteins will move faster/further in the gel than larger proteins and hence will progress more along the gel and will be closer to the anode.

Question 40

Give an example of a method of displacing bound proteins in affinity chromatography

Answer 40

High salt concentrations

Question 41

Explain the process of Western Blotting

Answer 41

The separated proteins from SDS-PAGE are transferred to a membrane using electrical current that moves the negatively charged proteins to the positively charged membrane. Then an excess of primary antibody is added to the membrane which specifically binds to the target protein. After unbound antibody is washed off an excess of a secondary antibody is then applied to the membrane. This secondary antibody binds selectively to the Fc region of the primary antibody. It also contains an enzyme bound it which when activated leads to the production of a fluorescent or coloured product.

Question 42

What gives the leucine zipper its name

Answer 42

Many of the hydrophobic residues that extend into the space shared between intertwined helices are leucines

Question 43

Explain the process of equilibrium sedimentation type density centrifugation

Answer 43

A steep sucrose gradient is established in a test tube with highest concentrations at the bottom. The cell contents are then added and centrifuged for a long time at a very high speed. This causes the organelles to deposit in the sucrose depending on their density with heavier, denser organelles depositing at the bottom of the tube and so on etc.

Question 44

3D radioactive peptide fingerprinting involves exposing peptides to radioactive phosphate or ATP and then X-ray radiation to determine regions of the peptide that are phosphorylatable, T or F

Answer 44

F – this is 2D radioactive peptide fingerprinting

Question 45

The supernatant produced in the final stage of differential centrifugation contains only viruses, ribosomes and large macromolecules, T or F

Answer 45

F – the final supernatant contains only pure cytosol

Question 46

What accounts for the high level of stability in leucine zipper domains

Answer 46

The hydrophobic leucine residues that extend into the space shared between intertwined helices are tightly packed together

Question 47

Often after separation based on isoelectric points, proteins are then separated using electrophoresis to distinguish proteins based on size, T or F

Answer 47

T

Question 48

Describe the process of gel filtration chromatography

Answer 48

Cytosol that has previously been centrifuged is loaded into a column containing a solid matrix of beads of a certain size. A neutral buffer and solvent is then added to the solution which acts to push the cytosol down the column. Larger proteins have a smaller volume to move through, due to their size, than smaller proteins. Hence larger proteins will leave the column first due to the sieving effect or the pore size between the beads. The different fractions of the cytosol can thus be collected drop-by-drop over time to separate the mixture.

Question 49

Extensions of the two leucine zipper helices straddle the minor groove of the DNA, T or F

Answer 49

F – they straddle the major groove

Question 50

Which class of amino acid residues extend outwards from each helix into the space shared between them

Answer 50

Hydrophobic side chains

Question 51

Which residue(s) are often acetylated by acetyltransferase enzymes

Answer 51

Lysine

Question 52

Explain the process of differential centrifugation

Answer 52

Differential centrifugation starts with a cell homogenate that is centrifuged at low speed. This creates a pellet contains the heaviest cytosolic components/organelle such as nuclei, whole cells and the cytoskeleton. The supernatant from this centrifugation is then separated and centrifuged again at medium speed and second pellet is produced this time containing mitochondria, lysosomes and peroxisomes. The second supernatant is the centrifuged again at high speed to produce a pellet containing microsomes and vesicles. This supernatant is then centrifuged in the next step at very high speed to produce a pellet containing viruses, ribosomes and large macromolecules. In the final step the supernatant from this stage is spun again at a very high speed for a very long time to produce a supernatant containing only pure cytosol

Question 53

How can proteins that interact with DNA be identified

Answer 53

Attach a specific DNA sequence of interest to beads contained within the column. Pass a nuclear extract through the column and proteins that bind to this specific DNA sequence will be retained in the column. Unbound protein can be removed by washing the column through with a buffer. Finally bound proteins can be eluted from the DNA using high salt concentrations