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MBB 324 > L13 > Flashcards

L13 Flashcards

1
Q

Draw an energy plot of the different protein folding states. Label what each state means. Slide 4

A 
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
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2
Q

Draw a diagram showing protein folding from a statistical view point

A

Slide 5
U = unfolded state (random coils)
M = molten globular state (partially folded, folding intermediate)
N = native state, most stable structure, folded state

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3
Q

Describe the experimental steps in the Denature/Renature experiment by Anfisen.

A
  1. Unfold RNaseA; 8M urea + BME
    - > Inactive enzyme
  2. Dialyze RNaseA To remove Denaturants
    - >Inactive enzyme
  3. Added trace amts. Of BME to get correct Disulfides
    - > Active enzyme
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4
Q

What are the 2 denaturants used in the denature/renature experiments and their mechanism of action?

A
  1. Beta-mercaptoethanol (BME)
    - > Breaks Disulfide bonds
  2. Urea
    - > Breaks H-bonds
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5
Q

What are the advantages of Ribnuclease A (RNase A)?

A
  1. Free
  2. Lots available
  3. PURE
  4. Enzyme activity assay
  5. small and soluble,
  6. Helices
  7. Sheet
  8. 4 disulfides
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6
Q

Why do we add trace amts. Of BME to get correct disulfides in the last step of the denature/renature experiment ?

A

Ribonuclease A has 8 cysteine residues. How many different ways can 4 disulfide bonds form using 8 cysteine residues?

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7
Q

Draw a diagram of % folded as a function of [urea] or temp

A

Slide 8

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8
Q

How can % folded be measured by?

A
  1. Activity assay
  2. NMR
  3. Fluoresence
  4. CD (circular dichroism)
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9
Q

What does the sigmoidal curve of % folded graph represent?

A

Sigmoidal curve reflects the cooperative nature of the unfolding process (many H-bonds/vdW-bonds)

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10
Q

What are the 3 obstacles to reaching the native state?

A
  1. Incorrect disulfide formation
  2. Incorrect Xaa-Pro isomer (cis/trans)
  3. Aggregation thru exposed hydrophobic surfaces during folding steps
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11
Q

Solution to Incorrect disulfide formation

A

Solution: Disulfide exchange reactions via protein disulifide isomerase (PDI) or can also occur in cytoplasm via glutathione/GSH (Healthy cells have >90% reduced)

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12
Q

Example of a PDI and its structure?

A

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

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13
Q

Solution ot incorrect Xaa-Pro isomer (cis/trans)

A

Solution: peptidyl proline cis-trans isomerase (PPI)

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14
Q

Example of a peptidyl proline cis-trans isomerase PPI?

A

Peptidyl-prolyl cis-trans isomerase Pin1 bound to non-natural peptide inhibitor (PDB-ID: 2Q5A)

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15
Q

Solution to aggregation thru hydrophobic surfaces during folding steps?

A

Solution: Chaperones and chaperonins, GroEL-GroES uses ATP to refold proteins

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16
Q

Example of chaperones and chaperonins?

A

GroEL-GroEs

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17
Q

What are the 2 types of INTRAmolecular chaperones? Describe them.

A
  1. Type I
    - N-terminal peptide (encoded in n-terminus)
    - Assists tertiary structure
  2. Type II
    - C-terminal peptide (encoded in c-terminus)
    - Assists quaternary structure
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18
Q

Slide 14

A

a

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19
Q

Other name for natively unstructured proteins

A

Natively (inherently, intrinsically) Unstructured (disordered, unfolded) Proteins

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20
Q

How common are natively unstructured proteins?

A

~ 10% of all proteins are fully disordered.

~ 40% of eukaryotic proteins have at least one long (>50 AAs) disordered loop

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21
Q

4 reasons why these proteins evolved to be unstructured?

A
  1. Permits specific binding with fast on/off-rate
  2. Provides specific binding without strong binding
  3. They can cover a large surface with few residues
  4. They can be removed quickly by proteases (regulated)
22
Q

What is the traditional protein structure paradigm that natively unstructured proteins challenge?

A

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

23
Q

Evidence that stability is NOT a required condition for fxn?

A
  • 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.
24
Q

Since AA sequence determines 3-D structure, AA sequence should also determine ______

A

lack of 3-D structure

25
Q

What are 4 commons sequence signatures of disordered proteins?

A
  1. low content of bulky hydrophobic AA: I, L, V, W, F, Y, and C.
  2. HIGH proportion of POLAR and charged AAs: E, K, R,
    - > Also enriched in: G, Q, S, P, and A
  3. LOW COMPLEXITY sequences, i.e. sequences with overrepresentation of a few residues.
    - > While low complexity sequences are a strong indication of disorder, the reverse is not necessarily true, that is, not all disordered proteins have low complexity sequences.
  4. Disordered proteins have a LOW content of predicted SECONDARY STRUCTURE.
26
Q

Once purified, what are 5 methods for identification of intrinsically unstructured proteins

A
  1. FOLDED proteins have a HIGH DENSITY (partial specific volume of 0.72-0.74 mL/g) and commensurately small radius of gyration. Hence, unfolded proteins can be detected by methods that are sensitive to molecular size, density or hydrodynamic drag, such as size exclusion chromatography, analytical ultracentrifugation, Small angle X-ray scattering (SAXS).
  2. Unfolded proteins are also characterized by their lack of secondary structure, as assessed by far-UV (170-250 nm) circular dichroism (esp. a pronounced minimum at ~200 nm) or infrared spectroscopy.
  3. Unfolded proteins have exposed backbone peptide groups exposed to solvent, so that they are readily cleaved by proteases,
  4. Unfolded proteins undergo rapid hydrogen-deuterium exchange and exhibit a small dispersion (<1 ppm) in their 1H amide chemical shifts as measured by NMR. (Folded proteins typically show dispersions as large as 5 ppm for the amide protons.)
  5. The primary method to obtain information on disordered regions of a protein is NMR spectroscopy.
27
Q

Why are regions of sequence missing in high-resolution crystal structures?

A
  • Many crystallographic structures have missing loops – that is, ranges of AAs w/no atomic coordinates in the model.
  • “gaps” in model are often thought to be ARTIFACTS of INADVERTENT disorder in the crystal.
  • In some cases, gaps may be alerting us to presence of intrinsically disordered loops in an otherwise folded protein.
28
Q

It’s very crowded in the cell Why don’t all the proteins aggregate and precipitate out of solution?

A
  1. Electrostatic repulsion limits interaction
29
Q

Slide 23

30
Q

Slide 24

31
Q

Protein misfolding

A

Protein quality control
Protein homeostasis
Protein recycling

32
Q

Protein Misfolding

A
  1. Change in structure

2. Causes fiber/filament formation

33
Q

Draw a flow chart of native to protofilaments

34
Q

Describe Amyloid-beta Precursor Protein (APP)

A

a large membrane protein in nerves.

Plays a role in neural growth and repair.

35
Q

Draw slide 27

36
Q

First enzyme to be discovered? Second to be crystallized?

37
Q

Enzyme crystals played an important role in showing what

A

Enzymes were proteins and that they had a defined structure

38
Q

Under what conditions does pepsin work the best?

A

Strong hydrochloric acid

39
Q

Amyloid-B peptide has 2 _____ AA’s

40
Q

Amyloid fibres are ______ beta sheets

A

Antiparallel

41
Q

What does TSEs stand for?

A

Transmissible spongiform encephalopathies (TSEs)

42
Q

What are prions

A

Infectious proteins

43
Q

PrPc vs. PrPsc?

A 
PrP = cellular prion
PrPSc = Scrapie
44
Q

What is scrapie?

A

a fatal, degenerative disease that affects the NSs of sheep and goats. It is one of several transmissible spongiform encephalopathies (TSEs), which are related to bovine spongiform encephalopathy (BSE or “mad cow disease”) and chronic wasting disease of deer.

45
Q

Example of transmission spongiform encephalopathies

46
Q

What 3 types of proteins need to beremoved?

A
  1. Damaged or aggregated (miss-folded) proteins
  2. Metabolic proteins (enzymes)
  3. Regulation proteins (inhibitors, repressors, activators, cell cycle)
47
Q

What is the half-live of the following proteins:

Collagen						
Eye lens crystallin				
RFC1 (part of DNA polymerase)		
RPS8 (part of ribosome)				
Ornithine decarboxylase
A 
Collagen								117 years	
Eye lens crystallin						>70 years	
RFC1 (part of DNA polymerase)			9 hours	
RPS8 (part of ribosome)					3 hours	
Ornithine decarboxylase					11 minutes
48
Q

What is ubiquitin? What 2AA’s? How many of each

A
  • tag obsolete proteins for destruction highly conserved, found in almost all tissues (ubiquitous).
  • 2 lysine
  • 1 glycine
49
Q

Using a flow chart draw the process of ubiquitination

A

Slide 35
Ubiquitin

E1: ubiquitin-activating enzyme

E2/E3: ubiquitin-conjugating enzymes

Ubiquitin Ligase

50
Q

Describe proteasome

A
  • cell protein’s recycler
  • polyubiquitin attached to substrate protein interacts w/proteasome
  • 195 regulatory particle on top and bottom
  • 205 core (7 alpha 14 beta 7 alpha)
  • ATP used to unfold substrate protein

MBB 324 (17 decks)

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