What does the ribonuclease refolding experiment tell us?
Anfinson, 1972 Nobel Prize
- All info necess. for folding is in the protein’s primary sequence
- The environment of the cell is not necess. a requirement
What are some factors that can denature proteins?
- Heat
- pH (either extreme)
- Chemicals (org. solvent, urea, guanidinum, detergent)
Proteins fold into the _____________ state.
lowest energy
What is levinthal’s paradox?
It would take 10^87 years for a protein to find the lowest energy conformation by random sampling.
What are the two main folding pathways?
- secondary structure —> loops –> tertiary structure
2. hydrophobid aa’s condense —> secondary —> tertiary
Can all proteins fold on their own?
No. Chaperones!
What are the two main classes of chaperones?
- Heat Shock - Hsp70/Hsp40
2. Chaperonins - GroEL/GroES complexes
What are the bacterial homologous proteins of eukaryotic Hsp70/Hsp40?
DnaK & DnaJ
How do the Hsp70 / Hsp40 chaperones work?
at high temps they bind to hydrophobic region of unfolded protein & prevent aggregation
help some proteins cross membranes in their unfolded state.
How does the chaperonin GroEL/GroES complex work?
- unfolded protein binds to GroEL end without a GroES cap.
- GroEs cap binds the other side
- Protein is protected inside and can fold.
- protein is released after folding
What does protein disulfide isomerase (PDI) do to help protein folding?
Reduces incorrect disulfide bonds by breaking the incorrect ones and letting them reform correctly
How does Peptide Prolyl isomerase (PPI) work?
Helps proline in taking the cis conformation.
What are the three main protein purification methods?
- Gel filtration chromatography
- Ion exchange chromatography
- Affinity chromatography
How does gel filtration chromatography work?
Filters proteins by size.
larger proteins elute first, smaller proteins elute out last
How does Ion Exchange chromatography work?
Separates proteins based on charge.
Uses negatively charged beads to grab the positively charged proteins while the neg. charged proteins flow through the beads.
How does Affinity Chromatography work?
Filter soln. through column containing is preferred ligand (att. to the beads).
Then add lots of the ligand and the protein will follow it out of the column (release from the beads).
How can you evaluate protein purity?
Gel Electrophoresis
How does gel electrophoresis work?
Apply a charge to SDS polyacrylamide gel.
(stained) Molecules migrate down the charge.
Small molecules move faster & they are separated out by size.
How can we determine the mass of a protein?
Mass Spectrometry!
time of flight correlates to size and charge.
How can we determine the N-terminal side of a protein?
Edman degradation.
Add phenylisothiocnate –> covalently bonds to N-terminal aa.
Release the N-terminal aa. (lower pH) and identify it.
Rinse and repeat —> N-terminal sequencing!
What does Western Blot do?
- Separate proteins onto SDS polyacrylamide gel.
- Blot onto PVDF membrane
- React with antibody (Ig)
- Wash
- Detect anti-Ig coupled to enzyme
How is HIV detected?
Western Blot
What is the histological change in brains with prion disease?
Holes in the brain.
Alpha helices in normal prion —> beta sheets in abnormal prion
What is the Tm of a protein?
temperature where 50% of the protein is denatured.
higher Tm = more stable protein
