Protein Folding

Question 1

What are the characteristics of protein folding?

Answer 1

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

Question 2

Where does protein folding occur?

Answer 2

In the cytoplasm

Question 3

Why are proteins metastable?

Answer 3

They spontaneously unfold

Question 4

What did Anfinsen study?

Answer 4

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

Question 5

What is the structure of the 3D energy landscape?

Answer 5

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

Question 6

What is the Levinthal Paradox?

Answer 6

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

Question 7

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

Answer 7

Ranganathan

Question 8

How did Ranganathan observe the WW motif?

Answer 8

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

Question 9

What is the molten globule?

Answer 9

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

Question 10

Is protein denaturisation cooperative?

Answer 10

NO-No equilibrium is reached

Question 11

What variables are observed to define the folding pathway?

Answer 11

Structures of intermediates

Rate and energy barrier of each stage

Question 12

What is the structure of Lysozyme?

Answer 12

129 residue
globular
4 disulphide bonds
Mixed α/β fold

Question 13

How are lysosyme folding kinetics observed?

Answer 13

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

Question 14

What markers can be used during protein folding?

Answer 14

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

Question 15

What does ANS bind to?

Answer 15

Hydrophobic surfaces

Question 16

What wavelengths does Near UV CD measure?

Answer 16

240-300nm

Question 17

What does Far UV show?

Answer 17

secondary structure

Question 18

What residues are used for intrinsic fluorescence observation?

Answer 18

Trp and Tyr

Question 19

What is the folding pathway?

Answer 19

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

Question 20

Why is Intrinsic fluorescence not fully reliable?

Answer 20

6 residues but 2 are exposed so fluorescence is maintained

Question 21

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

Answer 21

2

Question 22

How can residue specific information be gained?

Answer 22

phi-value mutational analysis

HX NMR

Question 23

How is the folding pathway determined?

Answer 23

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

Question 24

Why is 2D NMR not much use?

Answer 24

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

Question 25

What does pulsed labelling NMR show?

Answer 25

exposed amide hydrogens are exchanged with water first | α domain folds first then β domain

Question 26

What is the sequence of pulsed labelling NMR?

Answer 26

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

Question 27

What proportion of lysozyme folding occurs via stable α intermediate?

Answer 27

75%

Question 28

Why do smaller proteins have a smoother landscape?

Answer 28

Fewer stable intermediates and fewer contacts needed to define the fold

Question 29

How do large proteins fold?

Answer 29

In domains via stable intermediates in multiple pathways

Question 30

What is the non-specific hydrophobic collapse known as?

Answer 30

The molten globule

Question 31

What are the bimolecular protein complexes?

Answer 31

Misfolds and aggregates

Question 32

How are the half lives of proteins increased?

Answer 32

disaggregation, folding, removal of damaged proteins

Question 33

What do molecular chaperones do?

Answer 33

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

Question 34

Do molecular chaperones fold proteins?

Answer 34

No, they are not enzymes

Question 35

Where are molecular chaperones found?

Answer 35

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

Question 36

What additional functions do chaperones have?

Answer 36

Function in uncoating of vesicles in endocytosis

Question 37

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

Answer 37

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

Question 38

What is the prokaryotic equivalent of HSP70?

Answer 38

DNA K

Question 39

What is the eukaryotic equivalent of GroEL?

Answer 39

HSP60

Question 40

What are HSPs?

Answer 40

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

Question 41

What is HSP90 involved in?

Answer 41

Raf1 signalling and steriod hormone receptors

Question 42

What is the structure of HSP90?

Answer 42

ATP binding induces a change in dimer open/closed structure

Question 43

Which HSP is least well understood?

Answer 43

HSP90

Question 44

What are HSP100 class?

Answer 44

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

Question 45

What is HSP70 structure?

Answer 45

70kDa monomer with substrate and ATPase domain

Question 46

What is the HSP70/40 cycle?

Answer 46

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

Question 47

What is the HSP70/40 rate limiting step?

Answer 47

ADP/ATP exchange

Question 48

Does HSP40 have an ATPase domain?

Answer 48

No

Question 49

Where is HSP70 found?

Answer 49

In eubacteria, archeabacteria, eukarya as a synergy

Question 50

Where are the eukaryotic type 1 HSPs found?

Answer 50

Mitochondria and chloroplasts

Question 51

Where are eukaryotic type 2 HSPs found?

Answer 51

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

Question 52

What is the structure of GroEL?

Answer 52

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

Question 53

When does GroES bind to GroEL?

Answer 53

In presence of ADP/ATP

Question 54

How is GroE observed?

Answer 54

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

Question 55

What is the GroE cycle?

Answer 55

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

Question 56

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

Answer 56

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

Question 57

What cooperativity is shown between the GroEL rings?

Answer 57

Negative

Question 58

How are cryo-EM molecules observed?

Answer 58

With a negative stain of heavy metal salts

Question 59

What has Xray crystallography shown about the GroEL structure?

Answer 59

Apical domain binds GroES Intermediate domain Equatorial domain for ATP

Question 60

What is the HSP60 structure in archea?

Answer 60

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

Question 61

What is the HSP60 type 1 in eukaryotes?

Answer 61

9/10 subunits with 8 fold symmetry | Tissue specific isoforms

Question 62

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

Answer 62

E386 - R197

Question 63

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

Answer 63

K80 - E386 - D83 to open channel