L13 Flashcards
(50 cards)
Draw an energy plot of the different protein folding states. Label what each state means. Slide 4
U = unfolded states (random coils) M = molten globular states (partially folded, folding intermediates) T = transition state (highest nrg state) N = native state, most stable structure, folded state
Draw a diagram showing protein folding from a statistical view point
Slide 5
U = unfolded state (random coils)
M = molten globular state (partially folded, folding intermediate)
N = native state, most stable structure, folded state
Describe the experimental steps in the Denature/Renature experiment by Anfisen.
- Unfold RNaseA; 8M urea + BME
- > Inactive enzyme - Dialyze RNaseA To remove Denaturants
- >Inactive enzyme - Added trace amts. Of BME to get correct Disulfides
- > Active enzyme
What are the 2 denaturants used in the denature/renature experiments and their mechanism of action?
- Beta-mercaptoethanol (BME)
- > Breaks Disulfide bonds - Urea
- > Breaks H-bonds
What are the advantages of Ribnuclease A (RNase A)?
- Free
- Lots available
- PURE
- Enzyme activity assay
- small and soluble,
- Helices
- Sheet
- 4 disulfides
Why do we add trace amts. Of BME to get correct disulfides in the last step of the denature/renature experiment ?
Ribonuclease A has 8 cysteine residues. How many different ways can 4 disulfide bonds form using 8 cysteine residues?
Draw a diagram of % folded as a function of [urea] or temp
Slide 8
How can % folded be measured by?
- Activity assay
- NMR
- Fluoresence
- CD (circular dichroism)
What does the sigmoidal curve of % folded graph represent?
Sigmoidal curve reflects the cooperative nature of the unfolding process (many H-bonds/vdW-bonds)
What are the 3 obstacles to reaching the native state?
- Incorrect disulfide formation
- Incorrect Xaa-Pro isomer (cis/trans)
- Aggregation thru exposed hydrophobic surfaces during folding steps
Solution to Incorrect disulfide formation
Solution: Disulfide exchange reactions via protein disulifide isomerase (PDI) or can also occur in cytoplasm via glutathione/GSH (Healthy cells have >90% reduced)
Example of a PDI and its structure?
Yeast PDI, PDB: 2b5e 1 chain containing 4 thioredoxin-like domains, N-terminal (blue) and C-terminal (red) Domains are the catalytic domains, have the sequence motif CGHC – redox disulfide
Solution ot incorrect Xaa-Pro isomer (cis/trans)
Solution: peptidyl proline cis-trans isomerase (PPI)
Example of a peptidyl proline cis-trans isomerase PPI?
Peptidyl-prolyl cis-trans isomerase Pin1 bound to non-natural peptide inhibitor (PDB-ID: 2Q5A)
Solution to aggregation thru hydrophobic surfaces during folding steps?
Solution: Chaperones and chaperonins, GroEL-GroES uses ATP to refold proteins
Example of chaperones and chaperonins?
GroEL-GroEs
What are the 2 types of INTRAmolecular chaperones? Describe them.
- Type I
- N-terminal peptide (encoded in n-terminus)
- Assists tertiary structure - Type II
- C-terminal peptide (encoded in c-terminus)
- Assists quaternary structure
Slide 14
a
Other name for natively unstructured proteins
Natively (inherently, intrinsically) Unstructured (disordered, unfolded) Proteins
How common are natively unstructured proteins?
~ 10% of all proteins are fully disordered.
~ 40% of eukaryotic proteins have at least one long (>50 AAs) disordered loop
4 reasons why these proteins evolved to be unstructured?
- Permits specific binding with fast on/off-rate
- Provides specific binding without strong binding
- They can cover a large surface with few residues
- They can be removed quickly by proteases (regulated)
What is the traditional protein structure paradigm that natively unstructured proteins challenge?
states that a specific well-defined structure is required for the correct fxn of a protein and that the structure defines the fxn of the protein
Evidence that stability is NOT a required condition for fxn?
- Many unstructured proteins undergo transitions to more ordered states upon binding to their targets.
- The coupled folding and binding may be local, involving only a few interacting residues, or it might involve an entire protein domain. It was recently shown that the coupled folding and binding allows the burial of a large SA that would only be possible for fully structured proteins if they were much larger.
- The ability of disordered proteins to bind, and thus to exert a fxn, shows that stability is not a required condition for fxn.
Since AA sequence determines 3-D structure, AA sequence should also determine ______
lack of 3-D structure
