____mg/mL protein
400mg/mL protein
Avg protein
- ___nm diameter
- ____ nm space btwn
Avg protein
- 6 nm diameter
- 7 nm space btwn
The different paths to the folded state is often show on a ___ shaped plot
funnel
Obstacles to reaching the native state
- Incorrect disulfide bond formation
- Incorrect Xaa-Proline isomer (cis/trans)
- Aggregation thru exposed hydrophobic surfaces during folding steps
Advantages of ribonuclease A
- Free
- Lots available
- Pure
- Enzyme activity assay
- Small and soluble
- Helices, sheets, 4 disulphides
Denature/renature experimental steps
- Unfold ribonuclease A; 8M urea and BME
- > Inactive enzyme - Dialyze ribonuclease A to remove denaturants
- > inactive enzyme - Added trace amts of BME to get correct disulfides
- > active enzyme
Role of chaperones/chaperonins
- Neither can actively fold proteins
- Chaperones and chaperonins may be constitutively expressed (always present) or stress-regulated (so called heat shock proteins) by heat, cold, starvation, poisoning, and other forms of stress can also activate their synthesis
Fxn of molecular chaperones
Prevent misfolding and aggregation, but they cannot unfold misfolded proteins.
Prokaryotic Hsp70s are often called
DnaK
What are the 2 domains of chaperone DnaK/Hsp70?
- ATPase domain
2. Peptide binding domain
2 fxns of Hsp70/chaperone DnaK
- Bind to newly synthesized proteins as they come from the ribosomes to prevent them from misfolding.
- Import of nascent proteins into organelles
Describe Hsp90
- Homodimer
- ATPase
- Acts together w/Hsp70 and several other chaperone proteins
What are 4 inhibitors of Hsp90 with antitumor effect?
- Geldanamycin
- Celastrol
- Radicicol
- Gedunin
Describe prefoldin
- 6 legged octopus
- coiled coil
- 2 top structures are beta barrel
Fxn of chaperonins
Actively unfold misfolded proteins, using energy from ATP-hydrolysis
Example of chaperonin
GroELGroES
Describe the mechanism of chaperonin GroELGroES
- Uses ATP to refold misfolded proteins
- Rotation of ring
Fxn of sHsp? Structure of sHsp? Example of sHsp?
- In the presence of substrate proteins small heat shock proteins (sHsp) form globular oligomers with 12–42 subunits
- Lens alpha-crystallin consists of 32 subunits, forming a globule with 190 Å outer diameter and a cavity of 100 Å.
- The most well-known sHsp are the crystallins found in our eye lenses
