Chapter 4: Chaperones

Question 1

Describe functions of molecular chaperones

Answer 1

1) Assist in protein folding
2) Help in large complex assembbly
3) Transport (“Holdase”)
4) Prevent aggregation/help in disaggregation
5) Assist in unfolding
6) Buffer that allows new protein functions to evolve (Hsp90)

Question 2

Where can chaperones be found

Answer 2

Cytosol, membranes, organelles and extracellular space -> where there are proteins, there are chaperones

Question 3

What does Hsp stand for

Answer 3

Heat-shock protein that belong to the family of stress-induced chaperones

Question 4

Why Eukaryotes have more chaperone families and more members

Answer 4

Due to increased organism complexity that requires more specialized chaperones

Question 5

Name most common chaperones

Answer 5

Chaperones that assist in de novo folding, such as Hsp70s and chaperonins

Question 6

What is the function of Trigger factor

Answer 6

It is bound to ribosome and interact with the emerging polypeptides near the ribosome exit to prevent its improper folding until C’ terminus is translated.

• Peptidyl prolyl isomerase
• Deletion is non-lethal
• Only occurs in bacteria
• Forms a pocket

Question 7

What amino acid can be found in cis conformation 10-30%?

Answer 7

Proline

Question 8

Function and mechanism of Peptidyl prolyl isomerase

Answer 8

It catalyzes proline cis-trans isomerization, where arginine forms H-bond with the backbone N of the proline, giving the peptide bond a single-bond character

Question 9

Three examples of Peptidyl prolyl isomerase

Answer 9

1) Cyclophilins
2) FKPB binding proteins (TF)
3) Parvulins

Question 10

NAC stands for

Answer 10

Nascent-polypeptide-associated complex

Question 11

Main function of NAC

Answer 11

Prevents association of the ribosome, to which it’s bound, with the ER membrane translocation machinery when no ER signal is present

Question 12

What happens when protein is targeted to the ER

Answer 12

NAC does not interact, whereas SRP binds the proteins and pauses translation until inserted into ER

Question 13

Structure of Hsp70

Answer 13

N’ terminus: ATPase-containing domain consisting of a-helices that acts as a lid
C’ terminus: polypeptide binding domain that consists of b-sandwich and hydrophobic loops that act as a contact site for polypeptide

Question 14

Function of Hsp40

Answer 14

Delivers nascent polypeptide chain to Hsp70 and accelerates ATP hydrolysis by locking ATPase in ADP state

Question 15

Describe open and closed Hsp70 conformations

Answer 15

Open: low affinity for the nascent chain and high on/off rates, ATP-bound
Closed: high affinity, low on/off rates, ADP bound

Question 16

Function of NEF

Answer 16

Nucleotide-exchange factor, catalyzes ADP release from Hsp70

Question 17

When does polypeptide exits Hsp70

Answer 17

Upon ATP binding

Question 18

Protein released from Hsp70 has these options

Answer 18

1) Fold: Kf > Kon > Kagg
2) Rebind to Hsp70: Kf < Kon > Kagg
3) Be transferred to chaperonin system
4) Aggregate: Kf < Kon < Kagg (occurs under stress and overloading of chaperones)

Question 19

Describe the principle of Isothermal Titration Calorimetry

Answer 19

It measures the heat evolved upon titration of ligand with increasing sample, allows to determine Kd, n and ∆H. Consists of sample and reference cell that are at adiabatic equilibrium, where cells are constantly heated to maintain equal temperature.

Question 20

What cofactors affect ATPase of Hsp70

Answer 20

Hsp40, Hip, Bag

Question 21

Describe two families of chaperonins

Answer 21

1) 7-fold symmetry and 14 subunits that make 2 stacked toroids, found in bacteria (GroEL/GroES), mitochondria (Hsp60/Hsp10) and chloroplast (Rubisco/Cpn20)
2) 8-fold symmetry, eukaryotic cytosol (CCT or TRiC)

Question 22

Structure of GroEL-GroES complex

Answer 22

Consists of 2 heptameric cis and trans rings connected by equatorial domains, GroES co-axially binds to the ATP-bound ring activating the complex

Question 23

Mechanism of GroEL-GroES complex function

Answer 23

Folding intermediate is recognized by apical domain of GroEL (trans), whereas GroES forces polypeptide into the cavity and changes conformation (cis), ATP hydrolyzes in cis ring and binds to trans ring together with polypeptide, release of polypeptide and GroES from cis ring -> two rings alternate

Question 24

Location of Hsp90

Answer 24

Cytosol and ER, essential for viability in both

Question 25

General functions of Hsp90

Answer 25

• Buffers effects of mutations
• Prevents aggregation of unfolded proteins
• Secures signalling pathways
• Refolds heat-denatured proteins

Question 26

Hsp90 structure

Answer 26

N’ terminus: ATPase
Middle: substrate binding
C’ terminus: always dimer

Question 27

Mechanism Hsp90 function

Answer 27

Upon ATP and polypeptide binding, lid closes and two subunits dimerize, folding occurs during hydrolysis which is regulated, substrate is released when hydrolysis is complete

Question 28

What happens if proteins aggregate

Answer 28

Dissolved by Hsp104 in yeast and directed for refolding;
Targeted to aggresome and removed by autophagy;
Degraded (Hsp40 has E3 ubiquitin ligase activity)

Question 29

Example of cell stress response

Answer 29

HSF-1 transcription is released from Hsp70 when a lot of proteins require its assistance (heat shock response), HSF-1 upregulates transcription of Hsp70 and binds back to Hsp70 when stress is removed

Question 30

Vicious circle of proteostasis decline

Answer 30

Upregulation of chaperones, UPS and autophagy fails, proteins misfold and aggregate, existing chaperones are directed to fix misfolded proteins, in the meantime leaving behind mutant (temperature-sensitive) proteins, which misfold and aggregate