Lecture 3 - Protein Folding

Question 1

How to water-soluble proteins fold

Answer 1

compact structures with nonpolar cores

Question 2

What does myoglobin do

Answer 2

• protein responsible for carrying oxygen in muscle tissue, containing a Heme group

Question 3

How many amino acids are in myoglobin

Answer 3

• polypeptide chain consists of 153 amino acids

Question 4

How is myoglobin organised

Answer 4

• 70 % of the main chain is folded into eight ⍺-helices.
• Porforin Ring, and Iron ion in Heme group

Question 5

What is protein folding driven by

Answer 5

tendency of hydrophobic residue is to be excluded from water giving a hydrophobic effect

This means that secondary structures are amphipathic (different Hydro faces), and NH and CO groups are paired in hydrophobic core (through H bonding).

Question 6

What are covalent bonds

Answer 6

– 2 atoms share electrons to fill a covalent shell – strongest

Question 7

What is ionic bonding

Answer 7

donation of an electron to form electrostatic attractive ions

Question 8

What are Van Der Waals forces

Answer 8

Temporary Dipole-Dipole interactions

Question 9

How is tertiary structure dictated

Answer 9

by side-chain reactions –
Positive and negative salt bridges between amino acid

Question 10

What do ribonuclease do

Answer 10

Degrade RNA

Question 11

Why are disulphide bonds rarely found in the cell

Answer 11

High glutathione concentrations

Question 12

What is a chaotropic agent

Answer 12

Breaks up hydrogen bonding

Question 13

What is a reducing agent

Answer 13

Causes reduction reactions

Question 14

What are native disulphide parings

Answer 14

contribute to the stabilization of the thermodynamically preferred structure

Question 15

How does Urea (chaotropic agent) convert denatured ribonuclease into an active rn

Answer 15

Urea prevents side chain interactions, and preventing correct interactions between amino acids to form proper shape, only disulfide bonds present

Question 16

How do reducing agent help turn a denatured ribonuclease into an active rn

Answer 16

When Reducing agent is added, weak disulfide bridges and incorrect bonds are broken, so only the strongest bonds remain

• This occurs as there is a decrease in free energy as the scrambled ribonucleases are converted to the stable, native conformation

Question 17

What is a glycocelated protein

Answer 17

Protein covered in sugars

Question 18

What is alternative splicing

Answer 18

exons from the same gene are joined in different combinations, leading to different, but related, mRNA transcripts

Question 19

What is proteome

Answer 19

• The entire complement of proteins expressed in a cell
• The set of proteins expressed under a specific condition

Question 20

What are some post-translational modifications a protein undergoes

Answer 20

1. Addition of small chemical groups
2. Amino Acid Processing
3. Addition of Complex Molecules
4. Addition of Small proteins

Question 21

How does Protein production occur to create a desired drug eg. insulin

Answer 21

• Plasmid contains gene of interest
• E. Coli cells are heat shocked at 42 degrees for 30 seconds, making outside membrane perforated
• Plasmid enters cell and transforms, proteins are translated and produced.
• This can then be purified and used eg. insulin

Question 22

How is a desired protein purified from the bacteria that created it

Answer 22

1. Transformation of cell (plasmid with gene of interest) (antibiotics will kill any cell that hasn’t taken up plasmid which is encoded with antibiotic resistance)
2. Selection
3. Cell Growth and Protein Production
4. Cell Lysis (Homogenization Sonication)
5. Chromatography – pull out desired protein
6. Dialysis – swap protein dissolved fluid for desired medium
7. SDS-PAGE – to check purity
8. Protein Assay – ensures protein is fit for purpose

Question 23

What is column chromatography

Answer 23

Used to separate soluble proteins based on differences in molecular characteristics

Question 24

What are some methods of purifying a product

Answer 24

• Gel Filtration – Separation depends on molecular size
• Ion Exchange – Separation depends on molecular charge
• Affinity Chromatography – Separation depends on specific binding interaction

Gel Filtration (Size exclusion chromatography – SEC) – separates proteins based on molecular size
Smaller proteins get trapped in polymer bead by entering aqueous spaces
Large molecules cannot enter beads do fall to the bottom
Ion exchange – Separates proteins based on differences between molecular charge
Negatively charged protein repulsed by resin, pushed to the bottom
Reverse for positive
Affinity Chromatography – Separates proteins based on specific binding interactions

Question 25

What is a SDS-PAGE

Answer 25

the migration of a protein only depends on size

Question 26

What are protein assays used for

Answer 26

Check activity of the protein/viability of product