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Flashcards in Lecture 3 Deck (53)
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1

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

SUMOylation

2

What does SDS-PAGE stand for

Sodium Diethyl Sulphate Poly-Acrylamide Gel Electrophoresis

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

T

4

Which two amino acids can be phosphorylated

Serine and threonine

5

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

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.

6

How can DNA footprinting be used to confirm DNA binding proteins

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.

7

Explain the main differences between equilibrium and velocity density centrifugation

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.

8

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

Via hydrogen bonding

9

Describe the process of affinity chromatography

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

10

Outline the typical methods used in protein purification

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.

11

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

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

12

What phenomena is affinity chromatography said to rely on

Tight interactions (enzyme-substrate binding)

13

How is the starting cell homogenate obtained in differential centrifugation

Blending, sonication or grinding with a pestle and mortar

14

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

+95Da

15

What is coupled mass spectrometry

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.

16

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

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

17

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

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

18

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

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

19

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

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.

20

Describe the process of ion exchange chromatography

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.

21

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

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

22

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

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.

23

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

T

24

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

Whole cells, nuclei and cytoskeleton

25

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

Proline residues in collagen are hydroxylated

26

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

Hexa-histidine (6xHis)

27

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

Boiling with mercaptoethanol breaks the disulphide bonds between cysteine residues

28

Explain the process of velocity sedimentation type density centrifugation

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.

29

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

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

30

What is the enzyme responsible for the ubiquitination of lysine residues

Ubiquitin ligase