Protein modifications

Question 1

Name 3 methods by which protein structure may be determined

Answer 1

• X-ray crystallography
• Nuclear magnetic resonance (NMR)
• cryo-electron microscopy (crys-EM)

Question 2

Describe the process by which X-ray crystallography is carried out

Answer 2

1. Protein is purified
2. Crystallisation
3. Freezing of crystal
4. Data collection
5. Measure diffraction
6. Visualise electron density
7. Build structure

Question 3

How does NMR reveal protein structure?

Answer 3

• Certain nuclei (H1, C13, N11, N15) are intrinsically magnetic, spinning causes magnetic moment when a magnetic field is applied
• Nuclear Overhauser Effect (NOE) shows information on protons in close proximity

Question 4

What is cryo-EM most useful for?

Answer 4

Looking at large structures (e.g a virus) however low resolution

Question 5

What is the function of post-translational protein modification?

Answer 5

Add extra layer of functionality/regulation

Question 6

Describe the process of proteolytic processing

Answer 6

1. Proproteins (zymogens) translated in an inactive form to ensure that enzymes do not get degraded
2. ‘pro’ sequence cleaved off by enteropeptidase
3. Some proteins translated with ‘pre’ signal sequence which targets them to correct cellular compartments

Question 7

How does phosphorylation occur?

Answer 7

• Covalent addition of phosphate group to serine, threonine and sometimes (rarely) tyrosine
• Added by kinases and removed by phosphatases

Question 8

What is phosphorylation important for?

Answer 8

Intracellular signalling

Question 9

Where does acetylation occur and what are its functions in each case?

Answer 9

N-terminal - synthesis, stability & locomotion of proteins
Amino group of lysine - reversible modification important in the regulation of proteins (e.g histones)

Question 10

What is glycolysation?

Answer 10

• The covalent addition of a carbohydrate moiety which is either N-linked (amino group of asparagine residues) or O-linked (hydroxyl group of serine/theonine)
• Mostly occurs through secretory pathway, produces cell membrane proteins

Question 11

What is acylation and what are its two types?

Answer 11

• Covalent addition of a fatty acid group
• Increases hydrophobicity, plays a role in membrane targetting and protein-protein interactions
• Most commonly: meristoylation and palmitoylation

Question 12

What is meristoylation?

Answer 12

• Addition of meristoyl group to amino-group of an N-terminal glycine with an amide bond

Question 13

What is palmitoylation?

Answer 13

Addition of palmitic acid to cysteine/serine/threonine via a thioester/ester bond

Question 14

What is prenylation?

Answer 14

• Covalent addition of prenyl group to C-terminal cysteine residue
• Increases hydrophobicity and play a role in membrane targetting and protein-protein interactions

Question 15

What is methylation?

Answer 15

• Covalent addition of methyl group to amino group of lysine and arganine residues, not reversible
• When present on histones can act epigenetically to repress/activate gene expression

Question 16

When are co-factors used for proteins?

Answer 16

• Required for enzymes to regulate activity

- Can be metal ions or small molecules

Question 17

What occurs in the oxidation of a protein?

Answer 17

• Covalent disulphide bond between 2 cysteine residues
• Either co/post-translational
• Reversible

Question 18

How do proteins interact with eachother? Provide an example

Answer 18

• Commonly mediated by tetratricorepeat domains folds

e. g Ribonuclease complexes with an inhibitor with femtomolar affinity

Question 19

What are the 4 common motifs/folds in protein-nucleic acid interactions?

Answer 19

1. zinc finger
2. leucine zipper
3. helix-turn-helix

Question 20

What are the properties of the Zn finger?

Answer 20

Zn ion coordinated by 2histidine and 2cysteine which binds to the major groove in DNA, with the + charge neutralising it. Can recognise 3 bases

Question 21

What is the helix-turn-helix?

Answer 21

2* helix joined by a short strand important for gene regulation. Alpha-helix is inserted into the major groove of DNA

Question 22

How does the leucine zipper function?

Answer 22

• Interacts with the major groove of DNA via basic residues of the N-terminus in order to neutralise the negative charge
• Leucine every 7-8 residues creates zipper (2 chains whcih dimerise to form an amphipathic helix)
• Hydrophobic side chains interact with eachother and polar chains interact with eachother

Question 23

Why do protein complexes with nucleic acids need to be very specific?

Answer 23

In order to ensure fidelity