Williamson Folding And Design

Question 1

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

Answer 1

Bovine ribonuclease A
1960s
Denaturant and reductant
Removal saw refolding

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

Question 2

Folding vs aggregation

Answer 2

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

Question 3

Ab initio prediction
Why is it tricky
Solution

Answer 3

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

Question 4

Thermodynamics of protein folding

Answer 4

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

Question 5

How many bonds broken to unfold?

Answer 5

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

Question 6

3 main entropic contributions

Answer 6

Conformational entropy (-)
Hydrophobic interactions (+, stop water ordering)
Disulphide bonds (+ stop disorder)

Question 7

1 main enthalpic contributions

Answer 7

Electrostatic interactions (large -)

Question 8

Folding/ unfolding curves

Answer 8

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

Question 9

Conformational stability from denaturation curves

Answer 9

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

Question 10

Levinthals paradox

Answer 10

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

Question 11

Folding energy landscape

Answer 11

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

Question 12

Methods for studying proteins folding

Answer 12

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

Question 13

Name the 5 protein theories

Answer 13

Anfinsen spontaneous refolding
Hydrophobic collapse- promote water disorder, entropic
Framework model
Nucleation growth
Jigsaw model

Question 14

Hydrophobic collapse

Answer 14

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

Question 15

Framework model

Answer 15

Secondary structures form independently

Then dock together

Question 16

Anfinsen spontaneous folding

Answer 16

Protein spontaneous

Eqm between folded and unfolded states

Question 17

Jigsaw model

Answer 17

Same puzzle can be formed from different starting pieces

So a number of different structures could form first

Question 18

Nucleation growth

Answer 18

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

Question 19

Trapping intermediates by disulphide bonds

Answer 19

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?

Question 20

BPTI folding pathway

Which pathways does this support?

Answer 20

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?

Question 21

Protein folding by mutagenesis studies

Answer 21

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

Question 22

Study of CI2 protein inhibitor by energy analysis

Which model does this support?

Answer 22

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

Nucleation CONDENSATION

Question 23

Folding of CI2 in terms of energy diagram

Answer 23

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

Question 24

Nucleation condensation model

What does CI2 give a model for?

Answer 24

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

Question 25

D2O exchange to show protein folding

Answer 25

``` 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 ```

Question 26

Native state HX

Answer 26

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

Question 27

HX instead measured by mass spec | What is the advantage of this?

Answer 27

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

Question 28

Mass spec HX pulse of ribonuclease H1 results | Which model did this support?

Answer 28

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

Question 29

Protein folding conclusion

Answer 29

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

Question 30

What can delay protein folding? | How did this influence folding pathway models?

Answer 30

``` Aggregation Proline isomers Disulphide bonds Non native hydrophobic clustering Partial heme misligation ``` Likely that these events were mistaken for multiple folding pathways

Question 31

So which models actually fit protein folding?

Answer 31

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

Question 32

Yamasaki 2013

Answer 32

``` 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 ```

Question 33

Proline isomerisation

Answer 33

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

Question 34

Double jump experiment

Answer 34

Cis protein denatured and refolded with and without PPI | Enzyme increases folding rate

Question 35

Disulphide bond formation

Answer 35

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)

Question 36

Scrambled ribonuclease assay | Where is PDI found?

Answer 36

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

Question 37

Structure of PDI

Answer 37

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

Question 38

How is in vivo folding different to in vitro?

Answer 38

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

Question 39

Macro molecular crowding

Answer 39

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

Question 40

Co translational folding

Answer 40

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

Question 41

Molecular chaperones

Answer 41

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

Question 42

The 3 pathways of chaperone folding

Answer 42

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

Question 43

Trigger factor | Domains

Answer 43

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

Question 44

Hsp70 Function Structure

Answer 44

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

Question 45

Hsp70 cycle

Answer 45

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

Question 46

Experiment to determine hsp70 specificity

Answer 46

``` 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 ```

Question 47

GroEL/ES

Answer 47

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

Question 48

3 domains of groEL

Answer 48

L234, L337, V259 L263 V264 Make hydrophobic patch Polar residues moved to surface during conformational change Apical domains moves up Intermediate and Equatorial

Question 49

Why design new proteins?

Answer 49

New and novel catalytic activities Improve existing New structures

Question 50

3 general methods to design new proteins

Answer 50

Rational design Directed evolution Catalytic antibodies

Question 51

Rational design

Answer 51

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

Question 52

Modifying existing site

Answer 52

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

Question 53

De novo design of new site on existing scaffold

Answer 53

``` 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 ```

Question 54

Kemp elimination Reaction 2 designs

Answer 54

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

Question 55

3 aims of creating kemp enzyme

Answer 55

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

Question 56

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

Answer 56

``` 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 ```

Question 57

Coiled coils for de novo design

Answer 57

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

Question 58

Directed evolution

Answer 58

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

Question 59

In vivo screen

Answer 59

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

Question 60

In vitro screen

Answer 60

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

Question 61

Smart libraries

Answer 61

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

Question 62

Combined rational design and direction evolution | Kemp reaction again

Answer 62

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

Question 63

Analysis of R7 mutant for the Kemp reaction | Combined rational design and directed evolution

Answer 63

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?

Question 64

Catalytic antibodies How they work How to make them

Answer 64

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

Question 65

Ester hydrolysis by Abzymes

Answer 65

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

Question 66

Chelatase mechanism by antibodies

Answer 66

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

Question 67

3 applications of novel antibodies

Answer 67

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

Question 68

How good are antibodies?

Answer 68

Best antibodies approach least efficient enzymes

Question 69

Limitations of Abzymes | Improvement strategies 3

Answer 69

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