Topic 1, Lecture 2

Question 1

What are protein families? Give an example.

Answer 1

Proteins that have similar tertiary structure, and, therefore, similar but not identical functions. For example, elastase and chymotrypsin both look very similar but not identical. They both function to break things down but work on different substrates.

Question 2

Describe bonds in protein folding.

Answer 2

Protein folding is held together by non-covalent bonds: electrostatic attractions, hydrogen bonds, or van der Waals interactions. Proteins very often will need to change and the sheer strength of covalent bonds make it difficult to do so.

Question 3

Describe backbone to backbone bonding.

Answer 3

Hydrogen bonds between atoms of two peptide bonds

Question 4

Describe backbone to side chain bonding.

Answer 4

Hydrogen bonds between atoms of a peptide bond and an amino acid side chain

Question 5

Describe side chain to side chain bonding. Give an example.

Answer 5

Hydrogen bond between two amino acid side chains. For example, the electrostatic bonds between a slightly negative and slightly positive R-group.

Question 6

Describe hydrophobic and hydrophilic interactions. Give the model example.

Answer 6

Polar side chains will be on the outside, while nonpolar will be on the inside. The Oil Drop Model is a good example. If a nonpolar molecule is on the outside, you can assume a protein folding problem.

Question 7

Describe the functions intrinsically disorder random coiling.

Answer 7

Binding- wraps and binds to proteins Signaling- random parts phosphorylates to activated/inactivated portions
Tethering- flexibility tethers two pieces together
Diffusion Barrier- fills space (no order) just stuffs

Question 8

What is a positive outcome of random coils?

Answer 8

Good for connecting two domains, allowing one protein to bind to another protein, often parts of proteins that are going to get phosphorylated

Question 9

What percent of all eukaryotic proteins can adopt structures that are intrinsically disordered?

Answer 9

25%

Question 10

Describe rotation concerning the amino acids.

Answer 10

There is no rotation around the peptide bone because those bonds are covalent bonds that keep the amino acids together. Rotation occurs around the alpha carbon.

Question 11

What are the two angles described by the Ramachandran plot and what are their measurements?

Answer 11

The angle between alpha carbon and amino group is phi (x-axis). The angle between alpha carbon and carboxy group is psi (y-axis). Beta Sheets: phi= -125, psi= +125. Alpha Helices: phi= -57, psi= -47

Question 12

What four examples were we given of things that denature proteins?

Answer 12

Heat, pH, Urea, and Beta-Mercaptoethanol

Question 13

How does heat denature proteins?

Answer 13

Heat breaks noncovalent bonds, i.e. more movement=greater energy

Question 14

How does pH denature proteins?

Answer 14

pH changes the charges on side chains or basic and acidic amino acids, breaks ionic attractions

Question 15

How does urea denature proteins?

Answer 15

Urea break hydrogen bonds that held protein together bc there is water in urea

Question 16

How does beta-mercaptoethanol denature proteins?

Answer 16

Beta-mercaptoethanol is a powerful antioxidant that breaks disulfide bonds, specifically in cysteine

Question 17

If all denaturants are reversed, will the protein refold? Will it be the same?

Answer 17

The protein will refold and it will be the same because we don’t care about statistics.

Question 18

Define conservative substitutions.

Answer 18

Similar amino acids changes

Question 19

Define entropy.

Answer 19

The measure of disorder or randomness in a system

Question 20

Describe the energetics of protein folding.

Answer 20

Proteins will fold in coherence with the second law of thermodynamics because it takes more energy to order the water molecules around nonpolar regions of the protein than it does to move the nonpolar side chains inside the polar regions.

Question 21

Define denaturation.

Answer 21

The disruption of noncovalent interactions that stabilize the native conformation of a protein: heat, pH extremes, and chemical reagents.

Question 22

What is the difference between folding in vitro vs folding in vivo?

Answer 22

In vitro folding usually takes hours, while in vivo folding happens within minutes

Question 23

Define chaperones.

Answer 23

Chaperons are a class of proteins involved in the protein folding mechanism of cells. Many molecular chaperones are heat shock proteins.

Question 24

What are heat shock proteins and what do they do?

Answer 24

Heat shock proteins are examples of a large group of chaperones. They respond to heat extremes and function as a result of proteins being denatured.

Question 25

What are the examples of Hsp’s given in lecture?

Answer 25

Hsp 70, Hsp 60, Hsp 40

Question 26

Where does Hsp 70 work?

Answer 26

In the cytosol

Question 27

How does Hsp 70 work?

Answer 27

Hsp 70 binds to and stabilizes the unfolded or partially folded protein using ATP. This can prevent aggregation of unfolded or partly folded proteins. Hsp 70 acts co-translationally, which means that it works while the protein is being translated.

Question 28

How many types of Hsp 70 do humans have?

Answer 28

13

Question 29

Give the most detailed description of the Hsp 70 binding and acting process.

Answer 29

1. Hsp 70 binds transiently to nascent polypeptide. The open conformation of substrate bind domain of the Hsp 70 binds to the protein. The nucleotide binding domain binds to ATP.
2. ATP is hydrolyzed and Hsp 70 grabs tighter to nascent protein. ADP’s place is taken by ATP and Hsp 70 releases.

Question 30

Describe the accessory proteins in Hsp 70.

Answer 30

Accessory proteins DnaJ and Hsp 40 stimulate the hydrolysis of ATP which induces a conformational change in Hsp 70 that closes the SBD, tighter binding of Hsp 70, proper folding is facilitated.

Question 31

Describe Hsp 60 chaperonins.

Answer 31

The rim of the chaperon is hydrophobic. The proteins move inside to interact with the wall and refolds. The cylinder doesn’t know the protein is wrong. The only goal is to break the protein and refold it. The isolation chamber prevents aggregation of misfolded proteins. This works post-translationally, which means that everything happens after the protein has been fully formed. As ATP replaces ADP the CAP releases.

Question 32

Describe aggregated proteins.

Answer 32

Aggregated proteins gum up the cytoplasm (exposed nonpolar side chains)

Question 33

When can misfolding occur?

Answer 33

On-pathway, off-pathway, or irretrievably

Question 34

Discuss ubiquitin.

Answer 34

Ubiquitin gets rid of obsolete misfolded/unfolded proteins and damaged proteins. ALL misfolded proteins will have ubiquitin. It has an isopeptide bond.

Question 35

Define isopeptide bond.

Answer 35

An amide bond that is not present on the main chain of a protein, forms between the carboxy terminus of one protein and the amino group of a lysine residue on another (target) protein

Question 36

Describe the different ubiquitins.

Answer 36

Monoubiquitin- histone regulation, Multiubiquitin- endocytosis, Polyubiquitin- depends on lys #

Question 37

Describe the two cases of polyubiquitin.

Answer 37

Lys 48- proteasomal degradation, Lys 63- DNA repair

Question 38

List the steps to the activation of ubiquitin.

Answer 38

1. E1 (ubiquitin activating enzyme) accepts ubiquitin very sulfur bonding to carbon on carboxyl group of ubiquitin.
2. E2 (ubiquitin conjugating enzyme) is added to E1 that is already primed with ubiquitin.
3. Ubiquitin is now attached to E2

Question 39

List the steps that describe how ubiquitin ligase (E3) adds ubiquitin to target protein.

Answer 39

target protein binds to ubiquitin, and the target protein is polyubiquitylated for distruction. leaving you with ubiquitin ligase (E3)

Question 40

discuss intrinsically disordered (random coil) think intermediate sequence…

Answer 40

not apart of domain but still serves as a purpose to keep proteins together think about it being a rope…

Question 41

Under concept of intrinsically disorder (random coils), how does the cell signal?

Answer 41

serine, threonine, and tyrosine all have a hydroxyl group that can be replaced by a phosphate group.

Question 42

More energy to refold or unfold

Answer 42

unfold