Proteins and enzymes, Section 3, protein folding (Dr. Taylorson)

Question 1

What shows that primary structure influences protein folding ?

Answer 1

The fact that most proteins can fold and unfold in dilute solution w/o (most of the time) the help of other molecules.

Question 2

What are chaperones ?

Answer 2

Proteins that assist other proteins in their folding process.

Question 3

What is the native state of a protein ?

Answer 3

The native state of a protein (or nucleic acid) is when it is properly folded and assembled, thus is operational and functional.

Question 4

Consider a protein with only charged and polar AAs. Which structure would this protein adopt in the solvent (water) ?

Answer 4

If all the residues are polar or charged, they will be able to H-bond, as the polypeptide is being synthesized at the ribosome, with the surrounding water molecules. Thus, there will be no driving force and this molecule will not adopt any stable compact or regular shape.

Question 5

What is the main driving force for protein folding in solution ?

Answer 5

The hydrophobicity of certain R groups. When these groups are synthesized, the molecules cannot remain as an extended polymer.

Question 6

What is the hydrophobic effect ?

What does the consequence of this effect ?

Answer 6

The clustering of hydrophobic side chains from different parts of the same molecule.
This leads to the polypeptide becoming compact.

Question 7

Why is the hydrophobic effect thermodynamically favorable ?

Answer 7

Because it minimizes the total hydrophobic surface area in contact w/ the water and it brings the polarizable hydrophobic R groups together allowing Wan der Waals interactions to take place.

Question 8

How do polar amide groups dragged into the more hydrophobic interior of the protein satisfy their H-bonds ?

Answer 8

By finding 2ary structural elements w/ other main chain donors and acceptors.

Question 9

When can hydrophobic residues cluster of the surface of a protein ?

Answer 9

To form part of a specific binding site and compose a patch of mutually interacting non-polar groups.

Question 10

“The free energy of the folded state of the protein is much lower than the unfolded state.”

True or false ?

Answer 10

Actually, false. Protein folding is a thermodynamic compromise. There are many hundreds of interactions stabilizing a folded protein, and the difference in free NRG between the folded and unfolded state is generally not that large.

Question 11

What is denaturation ?

Answer 11

The loss of biological activity. It is evidenced by the unfolded state.

Question 12

How can proteins be denatured ?

Answer 12

By heating, to break the weak interactions that stabilize the protein.
By using denaturants such as Urea and Guanidinium hydrochloride; these compete for H-bonds of the polar groups of the backbone and side chain.
Detergents such as SDS (Sodium Dodecyl Sulfate) do a very similar thing.
Striped from its hydration shell, the protein loosens and water (or detergent) can enter the protein, causing it to burst.

Question 13

What dictates the stability of a protein ?

Give an example of unusually stable protein.

Answer 13

The compactness of its structure. Proteins isolated form thermophilic bacteria are unusually stable.

Question 14

What is tertiary structure ?

Answer 14

The final folded polypeptide chain held together by a number of mostly non-covalent forces in its most stable structure.

Question 15

What are the chemical interactions involved in tertiary structure ?

Answer 15

Disulfide bonds (0.22nm, 167kJ/mol)
Salt bridges (0.28nm, 12.5-17kJ/mol)
H-Bonds (0.30nm, 2-6kJ/mol)
Long range electrostatic interaction (variable, strong in non-polar regions, weak in water)
V der Waals inerations (0.35nm, 4-17kJ/mol in protein interior)
Metallic bonds… etc

Question 16

How are helical segments and beta-strands often connected ?

Answer 16

By beta-turns.

Question 17

What are loops ?

Answer 17

Stretches of AAS between 2ary structural elements.

Question 18

Where are loops usually found ?

Why is this important ?

Answer 18

At the surface of proteins and often protrude out into the solvent.
These are thus often involved in ligand binding, subpart recognition or membrane binding.

Question 19

Why can loop storage mutations more easily ?

What impact does this have on their role ?

Answer 19

Because they do not contribute much to the overall stability of the tertiary structure. It is often the loops regions that are involved in fct and their mutability provides a mechanism for molecular evolution in proteins.

Question 20

Can water molecules enter a polypeptide ?

How so ?

Answer 20

Some water molecules can be trapped inside the tertiary structure in internal cavities or in clefts/interfaces etc.
These water molecules are all part of the tertiary structure and may be important for the proteins functional activity.

Question 21

Why are proteins flexible ?

Answer 21

Because most of the forces stabilizing tertiary structure are non-covalent; they can break and reform easily.
Thus proteins are constantly fluctuating around an equilibrium structure. (Fluctuations can vary from 0.1nm to v large mvnt of a whole segment of the structure).

Question 22

Why is flexibility so important for proteins ?

Answer 22

Because an enzyme might change shape shape upon binding substrate, or a receptor when it binds its agonist etc.

Question 23

Are tertiary structures w/ disulfide bonds more stable than tertiary structure w/o ?

Answer 23

Yes.

Question 24

What proteins have disulfide bonds ?

Answer 24

Immunoglobulins, ribonuclease, insulin and pancreatic trypsin inhibitor.

Question 25

How often are metal ions founds in tertiary structures and which ions are usually found ?

Answer 25

Metal ions are found in 30% of all proteins. The bonding can be v tight or v loose. Water can also be involved.
The most common ions are calcium and zinc, but also potassium and sodium.

Question 26

What is the difference between stabilizing metal ions and the active site of metalloproteins ?

Answer 26

The answer is in the question. Stabilizing metal ions only have 1 function: providing stability.

Question 27

How do metal ions stabilize proteins ?

Answer 27

Metal ions often bind 4 or more ligands.

By binding several side chains, metal ions can act as multidentate cross-linking agents.

Question 28

What are cofactors ?

Answer 28

Cofactors are non-protein chemical compounds or metallic ions that are required for a protein’s biological activity to happen. These proteins are commonly enzymes.

Question 29

What is the cofactors of alanine aminotransferase ?
Of myoglobin ?
Cytochrome C ?
Polyamine oxidase ?

Answer 29

Alanine aminotranferase has a pyrodoxal phosphate bound to each subunit.
Myoglobin has a haem bound by a coordinate bond between histidine and the haem iron.
Cyt C has a haem attached in 2 places via a coordinate bond between the haem iron and a sulphur residue and the porphyrin ring of the haem.
Polyamine oxidase has PQQ (Pyrroloquinoline quinone) bound in 2 places in the centre of the protein.

Question 30

What are the different part of the proteins called ?

Why is this important ?

Answer 30

Domains.

These can have distinct roles: catalytic, binding, recognition, control, switching etc.

Question 31

What is quaternary structure ?

Answer 31

When the protein is composed of more than 1 polypeptide, they are said to be oligomeric and exhibit quaternary structure.
Individual subunits = monomers

Question 32

How are quaternary structures named ?

Answer 32

Homo/hetero to indicate if subunits are identical are different
Monomeric/dimeric/trimeric/tetrameric to indicate nb of subunits

Question 33

How fast are catalytic events compared w/ binding or signaling events ?

Answer 33

Catalytic events are fats and involve small scale motions, whereas binding events and signalling are often associated with slow, large scale mvnts.

Question 34

Give an example of an enzyme that undergoes a conformational change upon the binding of its substrate.

Answer 34

Triosephosphate isomerase.
An 8 residue loop is in the open conformation prior to binding, and closed down over the bound substrate to exclude solvent upon binding.