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Proteins > Protein Folding > Flashcards

Protein Folding Flashcards

1
Q

What are the characteristics of protein folding?

A

High fidelity
Dynamic
Subject to degradation
Be able to bind ligands tightly and specifically

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2
Q

Where does protein folding occur?

A

In the cytoplasm

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3
Q

Why are proteins metastable?

A

They spontaneously unfold

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4
Q

What did Anfinsen study?

A

NMR and Xray crystallography structures using reversible unfolding by pH/temperature/chaotropes

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5
Q

What is the structure of the 3D energy landscape?

A

Cone with high entropy, high entropy nascent chains at the top
Rugged conversions between intermediates
Native state with high stability at base

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6
Q

What is the Levinthal Paradox?

A

Time to search all conformations is much greater than the amount of time folding takes

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7
Q

Who observed the key 8 residues in the WW poly(pro) binding domain?

A

Ranganathan

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8
Q

How did Ranganathan observe the WW motif?

A

Using coevolution and conservation of residues for function in 100 proteins observed in phage binding assays

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9
Q

What is the molten globule?

A

A partially denatured structure with 2’ structure but not 3’ structure and a 10% volume increase

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10
Q

Is protein denaturisation cooperative?

A

NO-No equilibrium is reached

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11
Q

What variables are observed to define the folding pathway?

A

Structures of intermediates

Rate and energy barrier of each stage

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12
Q

What is the structure of Lysozyme?

A

129 residue
globular
4 disulphide bonds
Mixed α/β fold

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13
Q

How are lysosyme folding kinetics observed?

A

Denatured in 6M guadinium then diluted through stopped flow spectroscopy and measured changes in observable markers

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14
Q

What markers can be used during protein folding?

A 
HX-NMR
ANS binding 
Inhibitor binding to active site
Aromatic absorbances
Intrinsic fluorescence
Real time NMR
Near UV CD 
Far UV CD
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15
Q

What does ANS bind to?

A

Hydrophobic surfaces

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16
Q

What wavelengths does Near UV CD measure?

A

240-300nm

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17
Q

What does Far UV show?

A

secondary structure

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18
Q

What residues are used for intrinsic fluorescence observation?

A

Trp and Tyr

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19
Q

What is the folding pathway?

A

3ms hydrophobic collapse
10ms secondary structure
100ms persistant domains/near native
300ms native structure

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20
Q

Why is Intrinsic fluorescence not fully reliable?

A

6 residues but 2 are exposed so fluorescence is maintained

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21
Q

How many lysozyme intermediates has stopped flow fluorescence and NMR shown?

A

2

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22
Q

How can residue specific information be gained?

A

phi-value mutational analysis

HX NMR

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23
Q

How is the folding pathway determined?

A

Many methods, many timescales all give the same rates/folding pattern

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24
Q

Why is 2D NMR not much use?

A

Exchange takes up to 20 minutes which is too slow for folding

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25
Q

What does pulsed labelling NMR show?

A

exposed amide hydrogens are exchanged with water first

α domain folds first then β domain

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26
Q

What is the sequence of pulsed labelling NMR?

A

Denature
Partially fold to produce secondary structure
Quench at low pH to observe persistence/differential labelling

27
Q

What proportion of lysozyme folding occurs via stable α intermediate?

A

75%

28
Q

Why do smaller proteins have a smoother landscape?

A

Fewer stable intermediates and fewer contacts needed to define the fold

29
Q

How do large proteins fold?

A

In domains via stable intermediates in multiple pathways

30
Q

What is the non-specific hydrophobic collapse known as?

A

The molten globule

31
Q

What are the bimolecular protein complexes?

A

Misfolds and aggregates

32
Q

How are the half lives of proteins increased?

A

disaggregation, folding, removal of damaged proteins

33
Q

What do molecular chaperones do?

A

Bind unfolded proteins to reduce free concentration causing aggregation and misfolding without preventing folding

34
Q

Do molecular chaperones fold proteins?

A

No, they are not enzymes

35
Q

Where are molecular chaperones found?

A

Promiscuous and ubiquitous in all cells to bind any unfolded protein.
Can also be induced in stress

36
Q

What additional functions do chaperones have?

A

Function in uncoating of vesicles in endocytosis

37
Q

Which chaperones bind the nascent chain upon release from the ribosome?

A

Trigger factor and NAC bind in association with L23 ribosomal protein
HSP70 and HSP40 bind

38
Q

What is the prokaryotic equivalent of HSP70?

A

DNA K

39
Q

What is the eukaryotic equivalent of GroEL?

A

HSP60

40
Q

What are HSPs?

A

The simplest chaperones activated in times of stress and essential for all life

41
Q

What is HSP90 involved in?

A

Raf1 signalling and steriod hormone receptors

42
Q

What is the structure of HSP90?

A

ATP binding induces a change in dimer open/closed structure

43
Q

Which HSP is least well understood?

A

HSP90

44
Q

What are HSP100 class?

A

AAA+ ATPases involved in disaggregation or coupling to proteases for gated degradation

45
Q

What is HSP70 structure?

A

70kDa monomer with substrate and ATPase domain

46
Q

What is the HSP70/40 cycle?

A

HSP70 hydrolyses ATP to increase substrate affinity
HSP40 closes lid
GrpE exchanges ADP for ATP
Folding and substrate release

47
Q

What is the HSP70/40 rate limiting step?

A

ADP/ATP exchange

48
Q

Does HSP40 have an ATPase domain?

A

No

49
Q

Where is HSP70 found?

A

In eubacteria, archeabacteria, eukarya as a synergy

50
Q

Where are the eukaryotic type 1 HSPs found?

A

Mitochondria and chloroplasts

51
Q

Where are eukaryotic type 2 HSPs found?

A

In the cytosol as no HSP10 but a single peptide with a built in lid

52
Q

What is the structure of GroEL?

A

14 subunit as 2 rings of 7 monomeric 57.4kDa units with weak ATPase but high affinity

53
Q

When does GroES bind to GroEL?

A

In presence of ADP/ATP

54
Q

How is GroE observed?

A

Using cryo-EM with D398A mutant to slow hydrolysis for crystallisation of ATP bound form

55
Q

What is the GroE cycle?

A

7ATP bound to cis ring with positive cooperativity
Peptide bound in cis ring across 4 subunits
GroES cap binds to open cavity to 10A forming anfensin cage
Folding
ATP hydrolysis
7ATP and peptide bind in trans ring
GroES released
180’ rotation for trans to become cis

56
Q

How is cryo EM used to generate a 3D image?

A

Randomly tumbling protein produces lots of different 2D angles which can be combined into 3D structure

57
Q

What cooperativity is shown between the GroEL rings?

A

Negative

58
Q

How are cryo-EM molecules observed?

A

With a negative stain of heavy metal salts

59
Q

What has Xray crystallography shown about the GroEL structure?

A

Apical domain binds GroES
Intermediate domain
Equatorial domain for ATP

60
Q

What is the HSP60 structure in archea?

A

8/9 fold symmetry with 16-18 subunits made up of 1/2/3 types

61
Q

What is the HSP60 type 1 in eukaryotes?

A

9/10 subunits with 8 fold symmetry

Tissue specific isoforms

62
Q

Which residues are interacting in the GroEL T/inactive form without ATP?

A

E386 - R197

63
Q

Which residues are interacting in the GroEL R/active form?

A

K80 - E386 - D83 to open channel

Proteins (12 decks)

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