Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Proteins > Williamson Folding And Design > Flashcards

Williamson Folding And Design Flashcards

1
Q

Anfinsen experiment
What did this prove
Why don’t all refolding?

A

Bovine ribonuclease A
1960s
Denaturant and reductant
Removal saw refolding

Some can’t because of pro peptides etc.
Assistance chaperones- PDI, PPI (proline isomerisation)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Folding vs aggregation

A

Inclusion body formation
Eqm between folded and unfolded
Unfolded protein response with hydrophobic patches

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Ab initio prediction
Why is it tricky
Solution

A

The ‘protein folding problem’
Can we predict from sequence what it is going to do
Bioinformatics- Chou Fasman
Force fields for protein non covalent interactions too crude
Solution to thread sequences through known structures e.g. Phyre

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Thermodynamics of protein folding

A

G = H - TS
Enthalpy- making bonds is exothermic, large negative when folding
Entropy- will be less positive as the protein folds
For folding to be favourable, G needs to be negative to be spontaneous

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How many bonds broken to unfold?

A

About 1-5 bonds broken
This increases the positive entropy
Decreases the negative of the enthalpy
Means that delta G is less negative

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

3 main entropic contributions

A 
Conformational entropy (-)
Hydrophobic interactions (+, stop water ordering)
Disulphide bonds (+ stop disorder)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

1 main enthalpic contributions

A

Electrostatic interactions (large -)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Folding/ unfolding curves

A

Mid point of 4.5M urea
Transverse urea gradient electrophoresis
Unfolded state gives less migration during native page
Can also be done with CD, fluorescence, NMR shift change

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Conformational stability from denaturation curves

A

Keq is calculated for each point in the TS (U/F)
This then means that G can be calculated for each point
G = -RTln Keq
G is then plotted against denaturation
The Y intercept gives Gh2o which is the protein stability

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Levinthals paradox

A

Do proteins go through all conformations to reach lowest energy?
Only backbone, only phi and psi
2 conformations, only a and b in ramachandran
10^45 conformations
Would take 10^25 years for a protein to fold
The universe is 1.4 x10^10 yrs old

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Folding energy landscape

A

Experiments observed in distinct and different intermediates
Unfolded protein
Falls into molten globules as energy decreases
Multiple different intermediate pathways
Eventually reaches either a folded or unfolded low energy state

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Methods for studying proteins folding

A

Native page, sedimentation, size exclusion chromatography
Secondary by Far CD and D2O exchange
Tertiary by Fl, CD (near), NMR, ANS (dye to hydrophobic patch) and reverse phase HPLC

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Name the 5 protein theories

A 
Anfinsen spontaneous refolding
Hydrophobic collapse- promote water disorder, entropic
Framework model
Nucleation growth
Jigsaw model
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Hydrophobic collapse

A

The protein folds to hide its hydrophobic patches
Decreases the order of water around hydrophobic regions
Makes this entropic ally favourable
Would make the reaction more spontaneous

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Framework model

A

Secondary structures form independently

Then dock together

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Anfinsen spontaneous folding

A

Protein spontaneous

Eqm between folded and unfolded states

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Jigsaw model

A

Same puzzle can be formed from different starting pieces

So a number of different structures could form first

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Nucleation growth

A

Something initially folds
Rest of folding happens around the nucleation
Bit like crystal folding

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Trapping intermediates by disulphide bonds

A

BPTI has 3 S-S bonds
Can encourage folding by adding GSSG
Add iodoacetic acid at different points
This will iodoacetylate the free Cys residues
As time goes on, should be less iodoacetylation
Can analyse samples by ion exchange chromatography
Mass spec sequencing to see what order they fold in?

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

BPTI folding pathway

Which pathways does this support?

A

The peaks in absorbance of ion chromatography indicate the bonds forming
BPTI showed multiple folding pathways
14-38 bonds needs to be broken
Turned into 5-14 or 5-38 before -> 5-55
The 14-38 is then reformed last
Rate limiting step by isomerisation of bonds by PDI

Support for jigsaw?

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Protein folding by mutagenesis studies

A

E.g. Mutate residue in helix to see if in transition state
Measure free energy change
If not folding in TS, then no difference apart from final energy higher
If is in the TS, then both TS and end G will be higher
Calculated by change in TS/change in NS
If 1, then is in TS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Study of CI2 protein inhibitor by energy analysis

Which model does this support?

A

Showed folding directly from denatured state
Secondary and tertiary formed at same time
All elements partially formed at TS

Nucleation CONDENSATION

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Folding of CI2 in terms of energy diagram

A

Shown with A16, L49 and I57 in CI2 come together during folding
At the TS the entropic penalty rises fast than the enthalpic benefit
Means that at TS, G gets more positive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Nucleation condensation model

What does CI2 give a model for?

A

Weak local nucleus stabilised by a critical number of long range interactions
Large extended nucleus
Consolidation of the extended nucleus and structure occurs at the same time
CI2 a model for a FOLDON

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

D2O exchange to show protein folding

A 
Saturated with d2O at ph 6, all D
Folding by diluting in water
Increase to pH 10 - this speeds exchange
Only unfolded regions exchange
Returned to pH 6 to continue folding
HSQC spectra tuned for N, H
Less H folded quicker, %H plotted for each SS element
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Native state HX

A

Protein gradually denatured
At time points, proteins removed from denaturation and D exchange measured
Shows unfolding in stages
Each event is 2-state and cooperative
Series of smaller unfolding give a large unfolding
Each is a foldon

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

HX instead measured by mass spec

What is the advantage of this?

A

Protein Deuteration ph6
Left to fold for different time amounts
Then ph10 pulse
Unfolded will exchange

Gives QUENCHING ADVANTAGE pH 2.5 at 0 degrees
This can’t be done with NMR as a folded state is needed
Peptide digestion and ESI MS
H:D ration per peptide. Which parts of Seq folded first

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Mass spec HX pulse of ribonuclease H1 results

Which model did this support?

A

As time increases, time for folding before pulse increases
Peptides will be heavier
Those that show the quickest m/z increase have folded first
Allows low concs
Folding occurs in 2-state stepwise manner and cooperative

Showed sequential foldons, supported nucleation condensation

29
Q

Protein folding conclusion

A

First foldon random search
Foldons small to avoid levinthals paradox
First foldon is most stable, forms by NC
Large would face paradox, small foldons wouldn’t be able to overcome entropy barrier

30
Q

What can delay protein folding?

How did this influence folding pathway models?

A 
Aggregation
Proline isomers
Disulphide bonds
Non native hydrophobic clustering
Partial heme misligation

Likely that these events were mistaken for multiple folding pathways

31
Q

So which models actually fit protein folding?

A

Framework extreme example of foldons then docking
Some evidence of hydrophobic collapse to form first foldon
Nucleation condensation generally accepted

32
Q

Yamasaki 2013

A 
Temperature jump NMR
Heating and cooling system
46 -> 12 degrees
Scans over 12s
PCA 
Rate constant 0.2-0.7 s
Use to measure refolding
33
Q

Proline isomerisation

A

Prolyl peptide bond must be right
30% in cis form
Protein with one cis-Pro will show a fast and slow rate
Slow needs to be converted from trans first

34
Q

Double jump experiment

A

Cis protein denatured and refolded with and without PPI

Enzyme increases folding rate

35
Q

Disulphide bond formation

A

Active site for PDI has 2 Cys residues
Can bind to free Cys and catalyses bonds
This oxidises (joins) the protein bonds and reduces the PDI
Can also catalyse the reduction of a non native bond to replace it (e.g. BPTI pathway)

36
Q

Scrambled ribonuclease assay

Where is PDI found?

A

Denature ribonuclease with urea + reductant (DTT)
Oxidation and urea removal -> INACTIVE
Add more reductant to undo the bonds
Then add PDI
This time bonds reform in right places -> ACTIVE
converts RNA -> ribonucleotides
Increase in A260 nm
PDI found in ER as bonds are in secreted proteins

37
Q

Structure of PDI

A

Two a and two b domains
U shape
Cleft between alpha is active site
(a, a’, b, b’)

38
Q

How is in vivo folding different to in vitro?

A

1 mg/ml vs. 340
In the cells protein can form whilst on ribosome, different to denaturation experiments
Molecular chaperones

39
Q

Macro molecular crowding

A

4 different crowding agents on refolding on lysozyme
Drops refolding yield due to interference
Favours aggregation
Increases rate of hydrophobic collapse
Ovalbumin, BSA, Ficoll 70 and Dextran 70

40
Q

Co translational folding

A

Synthesis may be slower than folding
Ras and DHFR domains linked by linker region to be flexible
Measured Ras and DHFR activity

In vitro poor when fused
In e.coli less than 2% folding of fused protein
Rabbit reticulocyte lysate- 90% effective folding

Co translational folding prevents domain interference

41
Q

Molecular chaperones

A

Small hsps- no ATP use, may need help dissociating
Chaperones- hsp40, hsp70. Need ATP.
Chaperonins- hsp60, GroEL

42
Q

The 3 pathways of chaperone folding

A

Hsp70/60 independent- trigger factor protects
Hsp70 - shield hydrophobic patches
Hsp60- sequestered by groEL / groES

43
Q

Trigger factor

Domains

A

Unbound is a monomer or dimer
Interacts with folded proteins or vacant ribosomes
Association accelerated by hydrophobic patch
Might remain associated after its dissociation from ribosomal binding site

PPIase, C terminal and Ribosome binding
Open groove along whole protein to increase surface area
Liu et al- PPIase no activity, but assists folding of protein to crevice

44
Q

Hsp70
Function
Structure

A

Binds and stabilises nascent peptides
ATP for release
Protein may fold on release or be transferred to other protein
Important during heat shock response
Opening and closing by allosteric reactions upon ATP binding
NBD and peptide binding domain with alpha helical lid

45
Q

Hsp70 cycle

A

ATP increases affinity for nascent peptides
Hydrolysis closes lid
Undo incorrect folding to encourage native structure
Release of ADP is assisted by GrpE, Hsp110 etc.
Protein is either folded or unfolded

46
Q

Experiment to determine hsp70 specificity

A 
13 AAs peptide that overlap
Cross link to membrane
Add chaperone to bind
Wash
Western blot with antibodies
Mao the binding sites onto native protein- specific for buried sites
47
Q

GroEL/ES

A

2 heptamer rings make up GroEL
Binds protein by hydrophobic interactions
Binds of ATP causes elongation and opening of ring cavity
GroES lid binds
Replaced by hydrophilic surface, forces protein to fold by entropy
ATP hydrolysis
Affinity for groES lost, affinity of trans domain for protein lost
There is cooperative binding in the two chambers
90 degrees out of phase

48
Q

3 domains of groEL

A

L234, L337, V259 L263 V264
Make hydrophobic patch
Polar residues moved to surface during conformational change

Apical domains moves up
Intermediate and Equatorial

49
Q

Why design new proteins?

A

New and novel catalytic activities
Improve existing
New structures

50
Q

3 general methods to design new proteins

A

Rational design
Directed evolution
Catalytic antibodies

51
Q

Rational design

A

Prior knowledge
Modifies existing or
De novo- new site on existing scaffold or new scaffold

52
Q

Modifying existing site

A

Lactate dehydrogenase -> malate dehydrogenase
PPI converted into protease by inserting catalytic triad
Gave a good rate enhancement 10^8
But had a low Km value
Meant that Km/kcat wasn’t large, inefficient

53
Q

De novo design of new site on existing scaffold

A 
Making thioredoxin cleave PNPA
His catalytic nucleophile, less stable intermediate to rapid hydrolysis
Computational search of surface
Two mutants made:
F12H, Y70A the other contained L17H too
Very poor catalysts
54
Q

Kemp elimination
Reaction
2 designs

A

Ring opening of benzisoxazole by a base
Designed in silico
Glu base to abstract proton from carbon

1: Glu, Phe for pi stacking, Lys H bond donor
2: Asp:His dyad, Trp stacking and Ser

55
Q

3 aims of creating kemp enzyme

A

Base to abstract proton from carbon
Hydrogen bond donor to stabilise -ve charge on oxygen
Stabilisation of planar state by pi stacking

56
Q

Results of using TIM barrel for kemp reaction
2 designs
2 mutants designed to prove mechanism

A 
Big kcat/km is efficient
Mutant 1: 2.5 x10^5
2: 1.4 x10^5
Good enzyme is around 10^6
Further improved by direct evolution
E->Q mutant removes activity
Asp makes His better nucleophile, so removal lowers activity
57
Q

Coiled coils for de novo design

A

Self assembled cage particle
Coiled coils used as specificity between coils
Heterodimers and homotrimers
Folding driven by need to maximise coiled coil interactions
Sheet curves
Can attach enzymes etc. To inside or outside

58
Q

Directed evolution

A

No prior knowledge
Fitness landscape- islands of function within sequence space
Mutations to reach ‘peak’ fitness
Random mutagenesis induced by error prone PCR
Success dependent on screen or selection process
Need both for accuracy and high throughput
Better to screen for catalysis than substrate binding

59
Q

In vivo screen

A

dsRED
Fluorophore will slow half time
Plated and most red colony after 24h selected and rein put into PCR
Managed to get short half life, but the relative brightness decreased
E x Quantum yield
Shows that need to be conscious of other parameters changing

60
Q

In vitro screen

A

Protein of interest fused to g3p on protein surface
Gene for protein of interest in tandem with g3p to display
Easy to screen for activity to immobilised substrate
Isolate and amplify the phages that stick
Remove metal cofactors to allow binding but not catalysis e.g. Zn
Eluted by adding cofactor again

61
Q

Smart libraries

A

High throughout screens can’t always be done
Smaller libraries with high hit frequency
Targeting sites of substrate binding by mutations
But this needs prior knowledge
Saturation mutagenesis of these sites can be combined with cycles of random mutagenesis

62
Q

Combined rational design and direction evolution

Kemp reaction again

A

7 rounds of directed evolution
Catalyst with 200x increase in kcat/Km
Most effective was R7 mutant

63
Q

Analysis of R7 mutant for the Kemp reaction

Combined rational design and directed evolution

A

Catalytic E101 and K222 H donor not changed
Changes in adjacent residues for fine tuning
Some hydrophobic -> polar changes to hold K222 in correct position?

64
Q

Catalytic antibodies
How they work
How to make them

A

Abzymes
Only prior knowledge of mechanism needed
Lowers TS energy by stabilising, or destabilising substrate
Prepare antibody to haptenic resembling TS
Introduces strain
Covalently link haptene to carrier protein to induce Ab formation in B cells
B cells + myeloma -> hybridoma cell line

65
Q

Ester hydrolysis by Abzymes

A

Incoming OH around 0.6 A longer than TS
Needs to be stable enough
Not completely accurate
Success depends on how good the approximation is

66
Q

Chelatase mechanism by antibodies

A

Antibody raised against n-methylmesoporphyrin
The alkylation of the pyrrole nitrogen distorts the ring
Antibody has a Zn insertion rate similar to enzyme

67
Q

3 applications of novel antibodies

A

Novel catalysis of useful reactions e.g. Aldolase antibody for synthetic chemistry

Drug clearance- antibody for cocaine overdose, stable for 3 weeks, recycled unlike conventional antibodies

Pro drug activation- variation on ADEPT. Used instead of enzyme, avoids risk of immunogenicity which would limit repeat administrations.
One arm -> cancer antigen, other arm activates pro drug -> toxic drug

68
Q

How good are antibodies?

A

Best antibodies approach least efficient enzymes

69
Q

Limitations of Abzymes

Improvement strategies 3

A

Imperfect TSA
Lack of catalytic machinery
Rigid compared to enzymes
Mimics TSA for uncatalysed reaction

SDM to add catalytic residues, needs high res structure of complex
Direction evolution to improve catalytic efficiency
Hyper variable regions to increase flexibility

Proteins (8 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions