Lecture 7

Question 1

What is the Chou-Fasman method?

Answer 1

an empirical technique for the prediction of secondary structures in proteins

Question 2

Define Tertiary Strucutre

Answer 2

Tertiary Structure: spatial arrangement of AA residues that are far apart in the sequence an to the pattern of disulfide (S-S) bonds

Question 3

Give an example of a protein with tertiary structure

Answer 3

myoglobin

Question 4

State the function of Protein disulfide isomerase (PDI)

Answer 4

Protein disulfide isomerase (PDI) rearranges the polypeptide’s non-native S-S bonds into “correct” disulfide bonds

(this assumes that the disulfide bond that is present prior to the action of PDI is an “incorrect” disulfide bond (incorrectly folded))

Question 5

Protein Folding is a highley ______ process

Answer 5

cooperative

Question 6

Compare extracellular and intracellular proteins in terms of the amount of S-S bonds they feature

Answer 6

Extracellular proteins often have several S-S bonds

Intracellular proteins usually lack S-S bonds

Question 7

The protein folding and unfolding process is an “all or none” process. explain why

Answer 7

The molten globule stage that initiates folding is an intermediate stage that is very short

Question 8

When proteins are denatured, what level of organization is the first to be disassembled?

Answer 8

the tertiary structure

Question 9

What happens if there is only a partial loss of folding in a protein?

Answer 9

even a partial loss of folding structure will destabilize the remainder of the protein folding structure

Question 10

What is Quaternary Structure? give an example of a protein with quaternary structure?

Answer 10

Quaternary Structure: spatial arrangement of subunits and the nature of their interactions

The alpha2Beta2 tetramer of human hemoglobin

Question 11

State the 3 groups of Accessory proteins

Answer 11

PDI (Protein disulfide isomerases)

PPI (peptidyl prolyl cis-trans isomerases)

Molecular Chaperones (variety of molecules that serve to protect peptide structures)

Question 12

Describe what drives HSP 70 (HSP 40) and it’s function

Answer 12

ATP-driven

reverses misfolds ; newly synthesized proteins ; unfold/refold of trafficked proteins

Question 13

What is HSP 90 mainly used for?

Answer 13

the protection of signal transduction proteins

Question 14

What is significant about Nucleoplasmins?

Answer 14

they were the first molecular chaperone to be discovered

Question 15

Mitochondria contain their own heat shock protein molecules that are distinct from those that function in the cytosol. State these 2 HSP’s

Answer 15

HSP60 and HSP70

Question 16

State the 3 conditions and the 3 chemicals that are known to denature proteins

Answer 16

Conditions:
Heat

pH (extremes)

Agitation

Chemicals:
Detergents (SDS)

Chaotropic Agents (urea, guanidine hydrochloride)

Organic Solvents (TCA) [trichloroacetic acid]

Question 17

State the 5 methods of analysis that are used to determine if proteins are denatured or not

Answer 17

1. Turbidity
2. Circular Dichroism (CD)
3. UV Absorption
4. Fluorescence
5. Biological Activity

Question 18

What exactly is CD (Circular Dichroism) measuring in order to determine if proteins are denatured or not?

Answer 18

CD measures the differential absorption of right-handed and left-handed circularly polarized light resulting from the molecular asymmetry involving as chromophore group

(it is basically used to study the conformation of proteins in solution)

Question 19

What are molecular chaperones?

Answer 19

Molecular chaperones: essential proteins that bind to unfolded and partially folded polypeptide chains in order to prevent the improper association of exposed hydrophobic segments

Question 20

True or False:
Non-native folding, polypeptide aggregation, and precipitation are the only type of protein changes that can occur in the presence of molecular chaperones. explain.

Answer 20

False.

Non-native folding, polypeptide aggregation, and precipitation WILL NOT occur in the presence of molecular chaperones, preventing these is the main function of molecular chaperones

Question 21

Molecular chaperones allow misfolded proteins to refold into their ____ _____.

Answer 21

Native Conformation

Question 22

State the 2 major classes of Molecular chaperones and describe their functions

Answer 22

HSP’s: proteins that help repair any denaturation that may occur to a protein that is exposed to heat

Chaperonins: a protein folding container)

Question 23

Describe the 2 main HSP’s (describe them)

Answer 23

HSP70 family: coordinates cellular function by directing substrates for unfolding, disaggregation, refolding or degradation

HSP90 family: Integrates signalling functions, acting at a stage of folding of substrates

Question 24

State the 2 main Chaperonins

Answer 24

HSP60 proteins (GroEL)

HSP10 proteins (GroES) (Co-chaperone)

Question 25

Describe the structure of a chaperonin and outline the process by which it folds proteins

Answer 25

Chaperonins are a hollow cylinder with a cap on the top
(looks like a washing machine)

The protein enters, the cap closes and the cylindrical body changes shape to create a hydrophilic environment for the protein inside

The protein then folds, then the cap comes off, and a folded protein exits

Question 26

A number of pathological diseases are associated with protein aggregation. This is why ____ _____ is so tightly regulated

Answer 26

Protein turnover

Question 27

state the 2 main reasons why a cell would need to remove a protein

Answer 27

The protein is too old

The protein is damaged

Question 28

Name the main player in the protein homeostasis system that is known for degrading ubiquitinated proteins

Answer 28

proteasomes (a large protease complex)

Question 29

Describe the structure of the 26S proteasome

Answer 29

a 20S catalytic core with 2 19S regulatory units on the top and bottom of it

Question 30

Describe the functions of the 19S regulatory unit of the 26S proteasome (3 of them)

Answer 30

1. recognizes ubiquitinated proteins
2. isopeptidase cleaves off the ubiquitin
3. directs the protein into the catalytics core (20s)

Question 31

Describe the 20S regulatory unit of the 26S proteasome

Answer 31

It is a catalytic core composed of 28 subunits that form a “sealed barrel”

(access is controlled by 19S regulatory subunits on either side of it)

Question 32

Describe the process by which proteasomes and other proteases create free AA’s from ubiquitinated proteins.

Answer 32

Ubiquitinated proteins are processed into peptide fragments

peptide fragments are then further digested into free AA’s that can now be used for other biosynthetic reactions (recycling)