Post translational modification

Question 1

What can you learn from protein sequence comparison?

Answer 1

• insight into function
• signals for protein targeting
• post translational modifications
• internal repeats suggesting gene duplication
• info for producing antibodies or designing DNA probes

Question 2

What are the different methods of protein sequencing?

Answer 2

1. acidic or basic hydrolysis

2. edman degradation / direct sequencing

Question 3

How does acidic/ basic hydrolysis work?

Answer 3

1. breaking up the protein into its constituents
   - NaOH or HCl
2. Heated up
3. Allows the amino acids to be broken up
4. Mass spec or chromatography separates the AA out
   - can identify relative amounts of each a.a.

Question 4

What is edman degradation sequencing used for?

Answer 4

To identify the N/C terminus of a sequence

Question 5

How can you cleave an S-S bridge?

Answer 5

1. reduce the disulphide bonds
   - needs a reducer (DDT)
2. prevent the reformation by using a chemical agent
   - iodoacetate

Question 6

Where does Trypsin cut in an a.a. seq?

Answer 6

after Arg & Lys

Question 7

Where does Chymotrypsin cut in an a.a. seq?

Answer 7

after Phe, Tyr & Trp

Question 8

Where does Thermolysin cut in an a.a. seq?

Answer 8

Before Leu, Ile and Phe

Question 9

What are the roles of post translational modification?

Answer 9

• stabilisation
• modulation of activity
• signal transduction

Question 10

What are the different post translational modifications?

Answer 10

1. proteolysis
2. acetylation
3. protein-lipidation
4. glycosylation of proteins

Question 11

What occurs during proteolytic processing?

Answer 11

1. removal of N-terminal methionine
   - leads to blocked terminal residues
2. removal of hydrophobic signal sequences
   - signal peptides need to be cleaved when secreted (KDEL)
3. proteolytic activation of proproteins creating zymogens (inactive precursors)
   - inactive precursor enzyme but then the specific protease creates the active enzyme

Question 12

What is proteolysis?

Answer 12

cleavage via proteases

Question 13

State examples of where proteolytic activation of proproteins occurs?

Answer 13

Fibrinogen activation,
when cleaved, it forms fibrin, which is able to cause clots.
- fibrinogen in its inactive form can stay inactive and around the blood till clotting is needed
Regulation of Protein Kinase A
- dissociating the inhibiting holoenzyme R2C2 into a regulatory subunit R2 and 2 catalytically active subunits