1. Protein Folding BL

2. Protein folding is the translation of primary sequence information into secondary, tertiary and quaternary structural information
Don’t forget post-translational modifications. They change the chemical nature of the primary sequence and thus affect the final structure

5. other mutants... if vesicle targeting incorrect
the proteins are secreted by default

6. Michel Van Lun
Paul Berg (protein synthesis represented by dance)

7. Protein Folding Models
Hydrophobic collapse (oil drop model)
entropy driven, temperature dependent
size?
Nucleation theory
alpha helical and reverse turn
Viscosity/collision
Energy landscape

8. Folded intermediates "Molten globule"
When does a protein need help folding?

9. Protein Folding Machinery
When does protein folding need help?
During synthesis
Upon stress (esp. heat shock)
Players
Chaperone and chaperonins (heat shock factors)
Peptide binding proteins
ATP
Disulfide isomerase
Petidyl prolyl cis-trans isomerase

10. What happens if proteins don't fold properly?

12. Improperly folded proteins
Non-functional
improper interactions
nonproductive interactions
Aggregation
Targeted for proteolysis/degradation
Resource drain (commitment of material and energy)

13. Disulfide isomerization (PDI)
Protein disulfide isomerase
Human protein databank
Cysteine residues can be "tagged" with iodoacetate

14. Prolyl isomerase
Pin1 catalytic Lys residue "pulls" on the backbone carbonyl

15. Pin1 an important PPI Breast cancer:
Pin1 isomerizes phosphorylated Ser/Thr-Pro
Regulates various cellular processes.
Overexpressed in human breast cancer.
Overexpression causes upregulation of cyclin D1 and transformation of breast epithelial cells.
Alzheimer's Disease
Pin1 becomes depleted from the nucleus of diseased neurons
Redirected to the large amounts of abnormally phosphorylated tau proteins that will aggregate into filaments.
This depletion from the nucleus may ultimately contribute to neuronal cell death by reactivating the cell cycle.

16. What property do misfolded proteins share?
We have seen that protein/protein interactions can be highly specific
Chaperones and Chaperonins must bind a wide variety of different proteins
What is the mechanism of recognition that protein is misfolded?

17. Chaperones: Variations on a theme

18. DnaJ (Hsp40) Binds folding and misfolded proteins
Contains a 30aa glycine-rich region ('G' domain'), a central Zn binding domain containing 4 repeats of a CXXCXGXG ('CRR' domain) and a C-terminal region of 120 to 170 residues
Associates with DnaK
Discovered as requirement for viral replication

19. DnaJ
hydrophobic = yellow
charged = blue

20. DnaK (Hsp70) Binds folding and misfolded proteins Associates with DnaJ
ATP binding and hydrolysis
ER homologue Grp78
The interaction of DnaK-ATP to substrate proteins is transient and characterized by high on/off rates
2 domains
alpha helix
beta sheet:peptide binding

21. dnaJ, dnaK, grpE (foldosome)
Cytoplasmic (ER homologues)
Interaction between the J-domain of DnaJ and the N-terminal ATPase domain of DnaK stimulates ATP hydrolysis.
A second interaction mediated by zinc center II locks DnaK on substrate.
The nucleotide exchange factor GrpE then exchanges ADP with ATP and unlocks DnaK.

22. Chaperones

23. Chaperonin complex GroES/GroEL
Journal of Biochemistry (5):

24. GroEL (hsp60) Chaperonin
7-fold symmetry
two rings

25. GroEL (Hsp60)

26. GroEL

27. GroEL/GroES complex

28. GroEl/GroES complex Free GroEL binds denatured proteins very tightly.
GroEL undergoes an allosteric transition from its tight-binding state to a weaker binding state on the cooperative binding of nucleotides (ATP/ADP) and GroES.
GroES modulates binding of GroEL
7 ATP molecules bind to GroEL
ATP hydrolysis drives transition back to tight binding state

29. Folding cage/Iterative annealing
Denatured proteins bind to the resting GroEL·GroES·nucleotide complex.
Fast-folding proteins are ejected as native structures before ATP hydrolysis.
Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis.
Hydrolysis causes transient release of GroES and formation of the GroEL·denatured-protein complexes with higher annealing potential.
The intermediately fast-folding complex is formed on subsequent rebinding of GroES.
The ATPase activity of GroEL·GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster-folding R-states to the tighter-binding/stronger annealing T-states.

30. What happens when an Anfinsen-cage chaperone pulls its target?
Percolator model
What happens when an Anfinsen-cage chaperone pulls its target?
The initial volume of the cis cavity (assumed to be A3)
Water excluded from chamber on protein target
When ATP binds to the cis ring and induces a large conformational change increasing the cavity volume to A3
Water moves from the outside (organized structure) to the inside of chaperone and inside of target protein
Influx of water - resolvation of protein
10 to 15 kcal/mol - provided by the conformational changes driven by ATP-hydrolysis?

31. Thermosome (Archaea)

32. Hsp90 Abundant 2-3% eukaryotic cytoplasmic protein (also nuclear)
ER homologue grp94 glycoprotein folding
ATP binding and hydrolysis in both N- and C-terminal domains
Required for protein folding
Prevents protein aggregates
Deletion embryo lethal
ADP
Hsp90 N-terminal domain

33. Calnexin/Calreticulin (lectin chaperones)
Calnexin (CNX)/calreticulin (CNR) are ER chaperones and bind glycoproteins
No homologues in other compartments/Unique action
Calcium binding
CNX membrane anchored/CNR lumenal

34. Calnexin/Calreticulin
Repeated cycles of deglycosylation, chaperone binding and reglycosylation
Glucosyl transferase is sensor for incomplete folding
Removal of glucose on N-glycans is signal for correct folding/exit to Golgi
Removal of terminal mannose of middle branch of N-glycans is signal for folding failure. Mannose trimming of ER client glycans render them prone to degradation.

35. Osteosarcoma line
Blue Hoescht (DNA)
Red Cy3 anti CNX antibody (CNX/ER)
Green Phalloidin (Actin cystoskeleton)

36. CNX/CRT in Immune response
Calnexin binds with partially folded immunoglobulin proteins and retains them in the E.R. until they are completely folded
Recognizes partially glysosylated MHCI
Calreticulin retains MHCI until it binds peptides for display