Lecture 4: Protein Folding Flashcards
Forces holding proteins together
- Electrostatic attractions
- H-bonds
- Van der Waals forces
Hydrophobic effect
Hydrophobic areas clump together; for misfolded proteins with exposed hydrophobic areas, leads to aggregation. Also the basis of denaturation by detergents (SDS) and chaotropics
Levinthal paradox
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.
Chaperone proteins
Proteins that restrict the folding pathway by preventing inappropriate contact and limiting possible intermediates. Makes folding kinetically faster (doesn’t affect thermodynamics).
Hsp70
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.
Hsp60
2 sided molecular machine that uses hydrophobic protein binding sites and a GroES cap to fold fully translated protein.
Proteasomes
Protein complexes that recognize polyubiquitinated chains for processive protein digestion; made of 2 hexamer 19S “unfoldase” caps and a 4-heptamer-ring 20S core.
Functions of proteasome regions
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
Ubiquitination
Addition of ubiquitin to polypeptide chains as a signal for degradation
How is ubiquitin added to proteins?
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.
Protein folding compartments
- Cytoplasm
- Mitochondria
- ER
- Chloroplasts
Features of ER protein folding
- Site for all secreted and membrane protein folding after import into ER lumen
- Site of glycosylation and other post-translational modifications
- Contains oxidizing environment (e.g. for disulfide bonds) and quality control mechanisms
How are proteins imported into organelles?
Proteins must be unfolded before entering. Two methods:
1. Cotranslational translocation (ER)
2. Post-translational translocation (mt, chloroplasts)
Retrotranslocation
Process by which misfolded or incomplete proteins within the ER are returned to the cytoplasm for degradation.
Protein Disulfide Isomerase (PDI)
Oxidizing agent in ER for disulfide bond formation, gets reduced in process
Peptidyl-Prolyl Isomerase (PPI)
Accelerates rotation about peptidyl-prolyl bonds to assist folding
BiP chaperone activity
Hsp70 chaperone which binds Ab heavy chains and stays bound until the light chain binds. Forces retention in the ER until assembly is complete.
Unfolded Protein Response (UPR)
Upregulation of chaperones due to increased amount of misfolded protein within the ER
Protein aggregate properties
Very stable, insoluble, abnormal, large collections of proteins (hydrophobic effect). Tend to be protease resistant.
Cotranslational translocation
Associated ribosome translates protein straight into ER lumen and chaperones via protein conducting channel.
Post-translational translocation
After free ribosome translation, protein enters through protein conducting pores in unfolded form.
Proteasome structure
2x 19S hexamer caps (unfoldase) and 4x heptamer rings forming the 20S proteolytic core.
How does glycosylation function in protein folding?
Glycosylation acts as a marker for protein folding in the ER; assists in some way in association with protein folding/retention
