Lecture 4: Protein Folding

Question 1

Forces holding proteins together

Answer 1

1. Electrostatic attractions
2. H-bonds
3. Van der Waals forces

Question 2

Hydrophobic effect

Answer 2

Hydrophobic areas clump together; for misfolded proteins with exposed hydrophobic areas, leads to aggregation. Also the basis of denaturation by detergents (SDS) and chaotropics

Question 3

Levinthal paradox

Answer 3

Proteins fold extremely quickly despite the extremely vast amount of possible conformations; led to the funnel energy landscape theory, that proteins go through a series of “molten globule” intermediates.

Question 4

Chaperone proteins

Answer 4

Proteins that restrict the folding pathway by preventing inappropriate contact and limiting possible intermediates. Makes folding kinetically faster (doesn’t affect thermodynamics).

Question 5

Hsp70

Answer 5

Acts co-translationally and post-translationally using ATP along with co-chaperone molecules. Sometimes requires multiple cycles to complete folding. Contains ATP-binding domain and substrate binding clamp.

Question 6

Hsp60

Answer 6

2 sided molecular machine that uses hydrophobic protein binding sites and a GroES cap to fold fully translated protein.

Question 7

Proteasomes

Answer 7

Protein complexes that recognize polyubiquitinated chains for processive protein digestion; made of 2 hexamer 19S “unfoldase” caps and a 4-heptamer-ring 20S core.

Question 8

Functions of proteasome regions

Answer 8

19S cap: recognizes polyubiquitin tag and uses ATP to pull on substrate with ring strain. Cyclically denatures protein.
20S core: active protease sites within core processively degrades proteins to 7-8 AA peptides

Question 9

Ubiquitination

Answer 9

Addition of ubiquitin to polypeptide chains as a signal for degradation

Question 10

How is ubiquitin added to proteins?

Answer 10

Ubiquitin is first loaded by E1 onto ubiquitin ligase (E2/E3 complex), which recognizes denatured protein and repeatedly attaches ubiquitin to form a polyubiquitin chain.

Question 11

Protein folding compartments

Answer 11

1. Cytoplasm
2. Mitochondria
3. ER
4. Chloroplasts

Question 12

Features of ER protein folding

Answer 12

1. Site for all secreted and membrane protein folding after import into ER lumen
2. Site of glycosylation and other post-translational modifications
3. Contains oxidizing environment (e.g. for disulfide bonds) and quality control mechanisms

Question 13

How are proteins imported into organelles?

Answer 13

Proteins must be unfolded before entering. Two methods:
1. Cotranslational translocation (ER)
2. Post-translational translocation (mt, chloroplasts)

Question 14

Retrotranslocation

Answer 14

Process by which misfolded or incomplete proteins within the ER are returned to the cytoplasm for degradation.

Question 15

Protein Disulfide Isomerase (PDI)

Answer 15

Oxidizing agent in ER for disulfide bond formation, gets reduced in process

Question 16

Peptidyl-Prolyl Isomerase (PPI)

Answer 16

Accelerates rotation about peptidyl-prolyl bonds to assist folding

Question 17

BiP chaperone activity

Answer 17

Hsp70 chaperone which binds Ab heavy chains and stays bound until the light chain binds. Forces retention in the ER until assembly is complete.

Question 18

Unfolded Protein Response (UPR)

Answer 18

Upregulation of chaperones due to increased amount of misfolded protein within the ER

Question 19

Protein aggregate properties

Answer 19

Very stable, insoluble, abnormal, large collections of proteins (hydrophobic effect). Tend to be protease resistant.

Question 20

Cotranslational translocation

Answer 20

Associated ribosome translates protein straight into ER lumen and chaperones via protein conducting channel.

Question 21

Post-translational translocation

Answer 21

After free ribosome translation, protein enters through protein conducting pores in unfolded form.

Question 22

Proteasome structure

Answer 22

2x 19S hexamer caps (unfoldase) and 4x heptamer rings forming the 20S proteolytic core.

Question 23

How does glycosylation function in protein folding?

Answer 23

Glycosylation acts as a marker for protein folding in the ER; assists in some way in association with protein folding/retention