Module 2: V1 - V4

Question 1

Is there empty space inside a protein? Is water excluded from inside proteins?

Answer 1

no, proteins are compact

yes

Question 2

Where are polar side chains usually found on the protein?

Answer 2

exposed on the surface

Question 3

Where are nonpolar side chains usually found on the protein?

Answer 3

buried in the core

Question 4

Are disulfide bonds found buried in the core or on the surface of a protein?

Answer 4

buried in the core because disulfide bonds are nonpolar in an oxidised state
however under reducing conditions cysteine is exposed on the surface of the protein because it is polar in this state (however it will not form a disulfide bond)

Question 5

Where is glycine and proline usually found in a protein? Why does glycine do this and under what conditions can proline do this?

Answer 5

where the polypeptide chain is turning around and changing direction
glycine does this because it has no side chain and is really flexible
proline does this where turns are compatible with its backbone geometry (therefore much less proline residues are found in comparison to glycine residues)

Question 6

What forces drive protein folding?

Answer 6

electrostatic forces, van der Waals interactions, hydrogen bonds, hydrophobic interactions

Question 7

Is the folded state favoured over the unfolded state?

Answer 7

yes

Question 8

What is the folded state called?

Answer 8

the native state

Question 9

What is the unfolded state called?

Answer 9

denatured

Question 10

In solution, what will an unfolded polypeptide tend to do?

Answer 10

spontaneously fold up

Question 11

What are factors that will denature proteins?

Answer 11

pH, temperature and denaturants

Question 12

Protein folding is cooperative. What does this statement mean?

Answer 12

generally, if any part of a protein fold is disrupted interactions with the rest of the protein structure are disrupted and the remainder of the structure will be lost (there is no such thing as a half-folded protein)

Question 13

Is protein folding/unfolding reversible?

Answer 13

yes, under the appropriate conditions

Question 14

What must occur in order for a protein to refold correctly?

Answer 14

the protein must be allowed to refold first before subjected to oxidising conditions, otherwise the disulfide bonds would be formed in the wrong place resulting in a scrambled protein

Question 15

Do disulfide bonds ‘direct’ folding?

Answer 15

no, it is actually folding that directs disulfide bond formation

Question 16

What is the role of disulfide bonds in the overall stability of the protein?

Answer 16

increase the relative stability of the folded state over the unfolded state(s)

Question 17

Do proteins always fold spontaneously? Why?

Answer 17

not always, conditions in the cell can make folding slow or impossible
this is because of molecular crowding

Question 18

What is molecular crowding?

Answer 18

cells are highly concentrated solutions of proteins/nucleic acids/sugars/lipids etc. and as a result inappropriate interactions may occur with other molecules before the protein can fold

Question 19

Why is misfolding likely occur in a nascent polypeptide as it comes off the ribosome?

Answer 19

because the polypeptide chain grows by sequential addition of amino acid residues to the C-terminal end of the chain and misfolding occurs because the sequence is not complete

Question 20

How do chaperons help avoid misfolding?

Answer 20

chaperones assist by binding to unfolded/partially folded polypeptides and protecting them from misfolding

Question 21

Where do chaperones temporarily bind?

Answer 21

bind to exposed hydrophobic regions preventing them from interacting with the wrong partners: inappropriate interactions

Question 22

Do chaperones fold proteins?

Answer 22

no, they help avoid misfolding

Question 23

What is the purpose of multiple cycles of binding and release carried out by chaperones?

Answer 23

allows the protein to fold into the correct conformation

Question 24

What happens if the protein does not fold properly after multiple cycles of binding and release carried out by chaperones?

Answer 24

the protein will be sent off for degradation

Question 25

What are favourable interactions in proteins?

Answer 25

electrostatic interactions, london dispersion (van de Waals interactions), hydrogen bonds and the hydrophobic effect

Question 26

What are electrostatic interactions?

Answer 26

long-range interactions between permanently charged groups

Question 27

What is an example of an electrostatic interaction?

Answer 27

salt bridges (ionic interactions), especially those buried in the hydrophobic environment which strongly stabilise the protein

Question 28

What are van der Waals interactions?

Answer 28

medium-range weak attraction between all atoms which contributes significantly to the stability in the interior of the protein

Question 29

How do hydrogen bonds interact in a protein?

Answer 29

interaction of N-H and C=O of the peptide bond leads to local regular structures such as ɑ helices and β sheets

Question 30

What is the hydrophobic effect?

Answer 30

the release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy

Question 31

Why don’t molecules need net charge to form electrostatic interactions?

Answer 31

this is because dipoles can be formed as a result of charge separation across a molecule which leads to a “dipole moment” (based upon electronegativity)

Question 32

When does a hydrogen bond occur?

Answer 32

when two electronegative atoms compete for the same hydrogen atom

Question 33

What are components of the hydrogen bond?

Answer 33

the main component is electrostatic (dipole of D-H interacting with the partial negative charge on A)
in strong hydrogen bonds there is also a covalent component (based on transfer of electrons from A - H)

Question 34

The electrostatic, hydrogen bond, and van der Waals interactions between two polar groups in aqueous environment is not particularly favourable because comparable competing interactions are possible with water. Why then do proteins fold?

Answer 34

proteins also contain nonpolar groups and water is a poor solvent for nonpolar groups
(nonpolar groups cannot form hydrogen bond networks and prefer to react with other nonpolar groups)
this process is driven by entropy

Question 35

Why is a polypeptide chain more ordered when it folds?

Answer 35

when folded, less water is attached to hydrophobic regions as these regions are now joined together, leading to an increase in entropy as the number of free water molecules increases

Question 36

Does disulfide bonding direct folding or does folding direct disulfide bond formation? Discuss the evidence.

Answer 36

folding directs disulfide bond formation
this is because in order for a protein to fold correctly the protein must be allowed to refold first before subjected to oxidising conditions, otherwise the disulfide bonds would be formed in the wrong place resulting in a scrambled protein
therefore folding directs disulfide bond formation

Question 37

Ideally, protein folding is encoded in the amino acid sequence alone. Why then do cells have, and need chaperones?

Answer 37

this because the polypeptide chain grows by sequential addition of amino acid residues to the C-terminal end of the chain and misfolding is able to occur because the sequence is not complete
therefore, chaperones are required because they bind to unfolded/partially folded polypeptides and protect them from misfolding

Question 38

Based on what you know about amino acid side chains, devise an experiment to measure the thermal stability of a protein ~150 amino acids long.

Answer 38

expose the protein to differing temperatures and measure the proportion of unfolded vs folded proteins found in solution for each temperature
determine if there is a relationship between an increase in temperature and an increase in proportion of proteins which are unfolded over proteins which are folded