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Flashcards in Protein Folding 1 Deck (84):
1

How are proteins converted from polypeptide chains to an active form?

Protein folding.

2

What is the process of protein folding mediated by?

A complex energy landscape that directs the unfolded state conformations into the encoded native structure.

3

Why is it not possible to code a set of amino acids to produce a specific 3D structure?

Because the mechanism of how this works is not fully understood - unsure of how the 3D structure is encoded in amino acids.

4

What are intrinsically disordered proteins?

Proteins that manage to have biological function without having 3D structure.

5

How many eukaryotic proteins possess a significant level of structural disorder?

~30%

6

What can IDPs be associated with?

Neurodegenerative diseases.

7

Which neurodegenerative disease is alpha sinuclein associated with?

Parkinson's

8

Which neurodegenerative disease is AlphaBeta protein associated with?

Alzheimer's

9

Which neurodegenerative disease is Tau protein associated with?

Taupathies

10

Which neurodegenerative disease is Prion protein associated with?

Prion disease.

11

What other disease are IDPs associated with?

Cancer - P53.

12

Why is it interesting to study IDPs?

Can help target the diseases more specifically.

13

What allows the amino acid code rather than the DNA code to encode protein fold ?

It is much more complex than DNA (which only has 4 bases)

14

Who defined the general principle of protein folding?

Anfinsen.

15

What protein did Anfinsen use to define protein folding?

Ribonuclease A.

16

How many amino acids are there in ribonuclease A and how is it folded?

~120 amino acids
Folded via disulphide bridges - 8 cysteines that combine with a very specific pattern of 4 disulphide bonds.

17

What drives the protein to fold into its native structure?

because this state is the most stable

18

What defines the conformational of the native state?

The difference in free energy between the native and unfolded states - (Delta)G(NU).

19

What is chemical stability?

The stability by which a pattern of bonds between atoms is conserved.

20

What is the main stability that protein folding is focused on?

Conformational stability.

21

What is conformational stability?

Refers primarily to the ability of adopting a well defined conformation rather than random coil (unfolded) state.

22

What are the angles in the backbone of the amino acids?

Phi – the angle between alpha carbon and nitrogen.
Psi – the angel between the alpha carbon and carboxyl group.
(omega - peptide bond)

23

Which amino acids has the greatest available conformations?

Glycine

24

Give examples of things that result in chemical instability.

Deamination
Hydrolysis of peptide bonds
Oxidation (of Met at high temperatures)
Elimination/randomisation of disulphide bonds/

25

What is the energy change in protein folding?

Goes from high energy to low energy state.

26

What does protein need to have to be spontaneous?

A negative delta G

27

Which part of protein folding is not favourable?

Entropy - negative entropy when going from disordered to ordered state.

28

Which conditions influence protein folding strongly?

Temperature and pH

29

Why is protein affected by temperature and pH?

Proteins have been selected by evolution to be folded under very specific conditions.

30

What does optimum conditions for folding depend on?

The environment that the organisms live in - mesophile/thermophile/extremophile

31

What is the average stability of a protein?

5-10kcal/mol

32

Why is the average stability of a protein so low despite all the favourable covalent interactions?

It is a balance of the unfavourable interactions and destabilising ones.

33

Why is it advantageous for proteins to be minimally stable?

This means they can be degraded easily - would be too costly otherwise.
Also allows for dynamic conformational changes which may involve partial unfolding.

34

What is an example of covalent interaction in proteins?

Disulphide bonds - can occur inter/intra molecularly.

35

What is an example of inter molecular disulphide bond formation?

Antibody light and heavy chains.

36

Are disulphide bonds reversible?

Yes.

37

Which enzyme sometimes assists disulphide bond formation?

Protein disulphide isomerases.

38

What is the main driving force of protein folding (contributing towards compaction)?

The hydrophobic effect - proteins very compact because of this.

39

Which residues can be mutated in a protein without much effect on folding?

Surface residues.

40

Which is the most variable part of the protein?

The loops.

41

What type of mutations effect folding normally?

Internal residue mutations.

42

What does 'protein structure and folding is hierarchical' mean?

Sub domains fold independently of domains - then assemble to form domains.

43

Give an example of a protein that folds hierarchically?

GroEL.

44

Which protein is able to withstand many internal residue mutations?

T4 lysozyme

45

How does lysozyme withstand internal residue mutations?

Local shifts in packing.

46

What percentage of identity tends to mean identical protein fold?

>40%

47

What lower percentage of identity can also confer identical fold?

20%

48

Does identical fold mean identical functionality?

No - the number of folds is not infinite so identical folds need to give different functions.

49

What is an example of two proteins that have 50% identity but different folds?

GB1 and Rop - can be mutated to 80% identity and still fold differently.

50

What is required for a technique to measure protein folding?

Must give two different signals for folded and unfolded state.

51

How can folding being induced in the lab?

Temperature increase
pH extremes
Organic solvent
denaturing agent

52

What must be considered when denaturing protein using temperature?

Its origin - what is its optimum conditions.

53

Give an example of a denaturing agent.

Guanidinium hydrochloride (GuHCl).

54

Define melting point.

Point at which 50% of protein is folded and 50% is unfolded.

55

What has distinct signals in CD when absorbing circularly polarised light?

The three different states - alpha helices, beta sheets and random coil.

56

Define light.

A oscillating wave of electric and magnetic fields.

57

In CD which field of light are you concerned with

Electric field.

58

What are the different types of oscillations light can have?

Planar, circular or elliptic.

59

What are the different types of oscillations light can have in its electric field?

Planar, circular or elliptic.

60

How do you polarise light?

Oscillating the light in one specific plane.

61

Is circularly polarised light chiral?

Yes

62

What are the two kinds of circularly polarised light?

Left handed and right handed - chiral.

63

Which kinds of molecule absorb the two kinds of polarised light differently?

Chiral molecules

64

What is a good wavelength to measure a CD spectrum and why?

220nm - largest gap between alpha helices (-15) and random coil (1) - easy to follow transition.

65

What is a bad wavelength to measure CD at and why?

~204nm - this an isodichroic point
alpha signal = random coil, no transition to follow.

66

What is the sum of the two polarised lights before they hit the sample?

Linearly polarised light.

67

What happens is one form of light is completely absorbed by sample?

Circularly polarised light is produced

68

What happens if there is equal absorption of both types of light?

linearly polarised light is produced.

69

What happens if the two types are absorbed differently?

Elliptically polarised light is produced.

70

What does a modern CD spectrometer allow you to do?

Set angles of light you want.

71

Advantages of CD.

Fast and cheap and there are programs for deciphering results.

72

What makes protein folding so fast?

Protein fold is encoded in the amino acids.

73

Other than time, why would a protein not want to sample all conformations?

Some conformations are harmful to proteins - prevent protein reaching native fold or conformations can aggregate.

74

Define FRET efficiency.

How much the acceptor is being excited.

75

Describe protein ensemble.

Protein place in denaturing agent - water added in, diluting the denaturing agent and causes protein to refold due to conditions being below Tm. Can measure exponential phase of refolding to gain kinetic constant.

76

What do FRET and Protein ensemble not give you information about?

The transition state.

77

Is the transition state the highest energy state?

Yes.

78

What defines the kinetics from going between states?
D->TS->N

The energy gap between states corresponds to the kinetics required to interchange between them.

79

Describe the general principle of phi value analysis.

The mutation of residues using alanine scanning to determine which ones are already folded in the TS.

80

If you mutate a residue and get a phi value of 1 mean?

Transition state and native state have been disrupted - residue is already folded in TS.

81

If you mutate a residue and get a phi value of 0 mean?

TS is unaffected - amino acid not yet folded in transition state.

82

What did phi value analysis of the PDZ domain show?

Denatured (unfolded) state could go through two different TSs. One of these resulted in intermediate which caused aggregation.

83

What in the intermediate TS of PDZ domain folding caused aggregation?

Difference in N terminal beta hairpin - it was warped and so could not dock onto rest of protein.

84

What was it about the intermediate state of PDZ domain that allowed it to form?

It was very stable