Waltho Flashcards
(113 cards)
1
Q
What are the factors affecting protein stabilisation and folding?
A
- Non-covalent interactions
- Dielectric effect
- Hydrophobic effect
- Chemical denaturation
- Thermal denaturation
- Disulphide Bonding (covalent interactions)
2
Q
What methods are used to study protein folding in the presence of denaturants and the folding pathways of protein intermediates?
A
- fluorescence spectroscopy; backbone and sidechain CD; and NMR to study protein folding and unfolding.
- NMR to study intermediates via amide proton exchange
3
Q
How do chemical denaturants affect proteins:
A
- Hydrophobic effect
- Cooperativity
^^Disruption of the structure of water - Preferential binding to unfolded states
4
Q
What can be found from investigations using chemical denaturants on proteins?
A
- The M-values of states / intermediates
- Whether its a molten globule state
- Kinetic protein folding intermediates
- Major folding transition state
- the effects of mutations
5
Q
What are the different state proteins can have?
A
- Unfolded states
- Completely disordered states
- Molten globule states
- Folded states
- Kinetic protein folding intermediates
- Major folding transition state
6
Q
What can be learned about protein folding from studying disulphide proteins?
A
- How disulphides are formed in proteins + how are they detected
- The stabilities of disulphides
- Conclusions about the nature of the major transition state for folding
- Population distribution of disulphide bonds vs denaturant concentration
- Formation of essential off-pathway intermediates
- Existence of parallel folding pathways
7
Q
What are the key points to discuss when writing about the structure and assembly of amyloids:
A
- Formation of protein deposits with regular order (fibres)
- Organisation of amyloid fibres and their amyloid protein chains
- Difference in the structure of globular and amyloid states
- How is amyloid assembly triggered (protein destabilisation)
- The reactions of assembly and formed intermediates
8
Q
What is known about prion disease?
A
- Their organisation and structure
- Mutagenesis and stability of partially folded states
- Overall structure of prion fibrils
- Primary chain organisation of prion fibrils
- Size of the infectious unit
- Structure of infectious unit
9
Q
What is the dielectric effect? (water example)
A
As polypeptides have both amine and carboxylic groups, when dissolved in water the amines hydrogen bond with both the surrounding water and the carboxylate groups of adjacent polypeptides. The attraction between the amine’s hydrogen and proximal water pulls the water molecule closer, adding to the repulsive force between the amine and carboxylate (-ve oxygen vs - oxygen).
The dielectric affect describes the effect of the environment on an electrostatic intermolecular force of attraction.
10
Q
What would be meant by a high dielectric effect?
A
A high dielectric effect describes the environments disruptive effect on the electrostatic attraction between groups (e.g. weakening a force by water via increased repulsion)
11
Q
What environment would lead to a low dielectric effect (for peptides)?
A
Surrounding hydrocarbons -> won’t increases the repulsive forces, maintaining the strength of the hydrogen bonding between adjacent peptides.
12
Q
What rule determines the strength of an electrostatic force?
A
The Coulomb rule:
Energy = (Charge on first group * Charge on second group) / (Distance * Dielectric constant)
13
Q
What is a dielectric constant?
A
The the amount by which the energetic contribution of electrostatics is reduced. (How much does the environment reduce the strength of interaction)
14
Q
Why is the Coulomb rule not fully representative ?
A
The Coulomb rule assumes the charge exists at a single point, but the charges are delocalised throughout the molecule. The distribution of charges is affected by interacting groups covalently bound neighbours and resultant polarisation.
15
Q
What relationship does the dielectric rule describe?
A
The relationship between H-bonds and the polarity of the surrounding environment (important in the stability of folded proteins)
16
Q
Role of London Dispersion Forces in protein interactions: (van der waals)
A
Induced dipole-dipole interactions caused by simultaneous dipoles by non-uniform charge. These form spontaneous attraction. Conversely repulsion can also be caused by atoms in too close proximity, with their radii pushing away form eachother. These both lead to the interatomic distance curve.
17
Q
Why do attractive forces decrease over distance?
A
The interaction between induced dipoles (vdW) forces decrease as distance increases due to groups being no longer in proximity to interact.
18
Q
What is considered when studying the feasibility/ stability of folded protein states?
A
Not their absolute free energy, but the free energy relative to other possible states.
19
Q
What does the delta G value mean?
A
The favourability/ feasibility of the reaction / change in conformation
20
Q
What does the delta H value mean?
A
The change in favourable interactions forming the species
21
Q
What does the T delta S value mean?
A
Measures the change in the degeneracy of the two species.
22
Q
The Hydrophobic effect:
A
The property of water to interact with non-polar regions of molecules (similar to the dielectric effect). The water is affected by the non-polar groups and form tetrahedral conformations, as the hydrogen bonding water molecules are repelled by the hydrophobic group. As hydrophobic groups are non-polar this further strengthens the hydrogen bonding between water molecules and increases in rigidity. This forms a cavity around the methyl group, allowing it to more free rotate (increasing delta S).
23
Q
What is the impact of the hydrophobic effect on delta H?
A
Delta H increases due to the increases stability of the hydrogen bonds, but decreases because the water is not closely packed.
24
Q
How does the hydrophobic effect impact the conformational equilibria of protein folding?
A
In protein folding, it’s found this effect contributes to the stability of the folded state, by an advantageous delta S outweighing an increase in delta H.
25
How does the hydrophobic effect impact the conformational equilibria of protein binding?
In the binding of some hydrophobic molecules into cavities, the hydrophobic effect is found to aid binding by an advantageous delta H outweighing a negative delta S
26
What is the idea of entropic cooperatibity?
When considering the role of multiple interactions involved in the feasibility of conformation change, you are not able to simply add the delta G values for each interaction. This is because the interactions 'cooperate' and strengthen each other non linearly.
27
How does sidechain fluorescence report on the protein environment?
Side chains absorb UV light and fluoresce at different wavelengths than that absorbed, the wavelength and amount of fluorescence is then affected by the environment. (KEY Changes to wavelength and amount emitted dependent on environment)
28
How does the backbone circular dichroism spectra report on a proteins secondary structure?
Far CD spectra results from the amide groups of the protein backbone (and their chirality), these groups are sensitive to changes in ordering through hydrogen bonding, and-so the resultant spectra can be used to identify secondary structures like alpha helices and beta sheets.
29
How does side chain circular dichroism report on a proteins mobility?
Near UV CD spectra is produced based on aromatic side chains in chiral environments, (typically core of folded proteins), the asymmetry is rotationally averaged in unfolded states, causing the response to tend to zero (when mobile), whereas restriction (from folding _ interactions) leads to peaks
30
How can NMR be used to investigate a proteins environment, mobility and structure?
The chemical shifts of each NMR signal reflects its local environment.
The dispersion of the spectra reflects the folding of the protein, with great dispersion when folded due to more varied environments.
If there is motion in the side chains and backbone this may lead to the spectra looking 'simpler' / denoised -> with same number of signals.
31
How does sidechain fluorescence report on the protein environment? EXAMPLE
Tryptophan emits different colours under UV dependent on the hydrophobicity of the environment, either green in hydrophobic conditions, or orange when aqueous. This information informs us of the environment surrounding the residue.
32
What is dichroism?
Where light absorption changes for different directions of polarisation.
33
What is circular dichroism?
CD is the absorption of two diffeent types of circularly polarised light,
34
How is CD used in spectroscopy?
Circular Dichroism Spectroscopy uses both a right-handed circularly polarised wave, and left-handed circularly polarised wave, to produce a spectra representing a proteins secondary structure.
35
What are the three methods types of methods used to denature proteins?
Chemical Denaturants +
Temperature + pH
36
Role of temperature in denaturing proteins:
Proteins can unfold at both too high and too low temperatures, with these parameters often affected by the pH of the environment. Proteins can fold at either because the free energy of the unfolded state vs the folded state doesn't change linearly with temperature.
37
Chemical Denaturants in denaturing proteins:
Denaturants help solubilise hydrocarbon, making their unfolded stable in the aqueous environment, thus making the unfolded conformation more entropically feasible (by hydrophobic effect).
38
What is the 'm value' of Gibbs x denaturant curves?
The slope / gradient, determined by the number of hydrophobic sidechains are exposed upon unfolding. Alternatively known as 'hydrophobic burial'
39
Examples of chemical denaturants used in labs?
Guanidinium chloride and Urea
40
What can thermal denaturation be used to find, and how?
The enthalpy of unfolding can be found using differential scanning calorimetry, The heat capacity of the protein solution is measured across temperatures, when there's a peak in the specific heat capacity, the enthalpy change between states can be calculated
41
What measure of protein stability can be found from differential scanning calorimetry (DSC)?
Tm - the temperature the the peak of folding enthalpy change,
42
What are the generalisation of enthalpy and entropy between folded and unfolded states?
Folded states are enthalpically more favourable (lower / more negative) but more ordered and-so less entropically favourable. Unfolded states are the reverse.
43
pH as a protein denaturant:
extreme pH unfolds proteins, because their ionizable groups of sidechains are complemented by other polar groups in the native state, however are less tolerated when their ionisation states are changed.
44
What model described the unfolded structure of a protein?
The random coil model.
45
What are the assumptions of the random coil model?
In the backbone of a protein the only rotatable bonds are C-N (phi) and C-C (psi) bonds.
46
What is the random coil model?
The random coil model defines the unfolded state of a protein as a combination of multiple randomly coiled 'microstates' with random rotation across the backbone.
47
What evidence supports the random coil model?
- Fluorescence, CD and NMR measurements of unfolded protein are similar to those of small peptides
- Unfolded states have low density
- Large radius (X-ray scattering)
-
48
What is the key limitation of the random coil model?
The random coil often over extends proteins, predicting them to be longer than experimentally found to be.
49
What is often investigated to try to determine the mechanistic pathway of a protein's folding?
The intermediate states of the protein during folding.
50
What are the difficulties of studying protein folding intermediates?
They're much less stable than the folded and unfolded states, making up very small portion of sample.
Folding is fast and unimolecular, and-so can't be slowed down by altering concentrations. Therefore to investigate intermediates, the change in kinetics is observed as a function of a parameter (temp, denaturant concentration, etc.)
51
What are the strategies for investigating protein folding intermediates?
Protein fragments + Mild Denaturing Conditions
52
Protein Intermediates: fragments
Idea is to prevent complete protein (re)folding by removing some protein required for the final fold.
- If amino acids are successively removed from either terminus of proteins, eventually the folded state
will be disfavoured. The resulting non-native state will often have the properties of a molten globule
state
53
Protein Intermediates: Thermal
Close to the thermal (un)folding transition where delta G ~= 0, there is a 1:1 mixture folded and unfolded forms.
54
Protein Intermediates: Chemical Denaturant and NMR
Using Chemical denaturant guanidine chloride leads to the formation of states not native folded nor unfolded. These intermediate states were analysed using NMR to further confirm the changes of the environments in the intermediate compared to that of the native.
55
What type of state do intermediates often take?
Molten globule states
56
Properties of molten globule states:
- Larger than native folded protein (x-ray scattering and size exclusion chromatography)
- Protein core can be penetrated by solvent
The Core is hydrophobic and behaves similar to a liquid (low rigidity)
Freely rotating side chains (random-core like NMR and no near UV CD response.
-Spectrum of behaviours observed between proteins,
57
Denatured intermediate States:
Dynamic ensembles of rapidly interconverting species. Denatured states are randomly collapsed
58
What is a spin label?
An organic molecule with an unpaired electron used for NMR and CD. this causes NMR signal loss according to increasing distance from the label. Allows for selectivity observing an environment local to the labelled group.
59
Kinetic Intermediates of protein folding: What are the methods?
Measurements for characterising the kinetics of refolding and intermediate states is determined by calculating rates. Fluorescence, Far CD, near CD, NMR, and NMR exchange of hydrogens (via D2O)can all be used to find different pieces of information.
60
How can denaturant dependency be used to create a kinetic model of protein folding?
The denaturant dependence of folding and unfolding rates for the folded, unfolded, intermediate, and transition states are observed. Using these values derived from the graphs, their energy values can be found, and their sequential order using the 'm-values'. This is done by plotting (y-axis free energy against x-axis m-value). Using a Ln Kobs plot against concentration provides insight into the number of intermediates (number of turns / slope changes)
61
How do spin labels help identify intermediate states of protein folding using NMR?
Ordinarily in a native state (folded) its NMR spectra when labelled with a spin-label would produce a couple of distinct peaks denoting the local features. However signal loss can appear uniform, with no bias and no particular peaks forming (featureless), the protein is collapsing , however the residues involved are randomly distributed across the structure at random times. This is reflective on an unordered collapsed ensemble.
62
What is the major transition state?
A transient partially folded state of the protein, usually the most unstable, characterised by being the short lived product of the largest free-energy step in the protein folding.
63
What are the 3 factors that slow protein folding?
- Topology (takes time to organise residues)
- A state transition must occur to transfer from molten globule to (solid) folded state
- Desolvation of hydrocarbon (expelling trapped water molecules) -> requires input of free energy to overcome resultant hydrogen bonding
64
What is the rate determining step of protein folding?
The change in confirmation between the unfolded state and a transition state, or an intermediate state and the transition state. (rate determined by their difference in free energy)
65
What is the mutational approach to structure determination?
-Shorten hydrophobic sidechains through mutagenesis. E.g. if you take a sidechain and shorten it, the amount of hydrocarbon exposed in different species will differ.
Kinetics measurements of the mutant can then be compared to the wild type. Change in free energy of states, informs of which states the side chain contributes to stability (via interactions).
66
What is the NMR approach to finding the structure of a folding intermediate?
Amide hydrogens within the backbone and side chains can exchange with D2O at an intrinsic rate Kint. The rate of this exchange slowed by protection of groups from the solvent, this factor is the protection factor. The protection factor is caused by a combination of hydrogen bonding and hydrophobic burial. The protection factor can be equated to the energy difference of the closed and open forms, and compared to the free energy diagram of folding. If the open state is less stable than the intermediate state, the position of the hydrogen bonding within the intermediate state.
67
Guanidinium Chloride vs Urea: As chemical denaturants
Guanidinium chloride results in slightly higher thermodynamic stability, attributed the stabilising effect of its higher ionic strength.
68
Compact Disordered States:
Significantly differ from folded states (shown by NMR spectra). States of Intrinsic Disordered proteins -> non random coil -> secondary structure forms between unordered regions via interactions.
69
How are disulphide bonds formed?
Redox reaction or protein with thiol groups. Often a molecule that makes a strong er disulphide bond will reduce a molecule that'd make a weaker one. Usually glutathione (GSSG oxidised or GSH reduced).
70
What are the two methods of studying disulphide exchange reactions?
- Reducing the pH of the solution, drops the concentration of thiolate anions with cysteine residue (via protonation), slowing their activity and providing a window for analysis.
- Chemical trapping of thiols via reaction with iodoacetic acid or iodoacetamide. (permanent trap however bias can be introduced due to slow rate of trapping)
71
Reactions of disulphide formation:
2 reactions steps: Protein's thiolate anion attacks glutathione -> half a glutathione binds to the sulphur group in the protein. Another thiol within the protein attacks the newly form ed disulphide bond, displacing the glutathione.
72
Cystatin as an example of the effect of disulphide on protein stability?
When comparing the free energy plots of Cystatin (oxidised and reduced) against m-value -> It's found that energetically both the oxidised and reduced states are similar. The key difference is the final state of each share as similar M-value, however are energetically very different, with the reduced form having a less negative free energy change.
73
In the cystatin example what do the oxidised and reduced forms show?
As disulphide bonds formation is by oxidisation, when the solution is oxidising the protein will form its disulphide bonds, whereas in a reducing agent they won't form.
74
In the cystatin example what is significant by the different energy values of the final state of the reduced and oxidised variations?
The reduced form is less thermodynamically feasible, meaning the final state is less stable and forms a molten globule. The oxidised cystatin forms a fully folded state, due to its successful disulphide bonding.
75
What are one of the key observations / deductions from the observation of cystatin free energy?
Molten globule and fully folded states don't coexist for the same form (oxidised / reduced) and therefore the phase change between the two is not step of folding. Instead the phase change was dependent on disulphide bond formation. The less negative gibbs energy change of the reduced form demonstrates the desolvation of phase change is this proteins key transition state barrier.
76
BPTI in folding pathways: First Experiment Step
Oxidised glutathione added to the fully reduced BPTI -> the most dominant first disulphide bond made is between 30-51 -> if this was random, this wouldn't be the case, as the most dominant would occur between the two most proximal residues. -> The 30-51 dominates the population because the formation of the bond leads to a change in native structure that stabilises the bond -> making the bond more difficult to break (altering equilibrium)
A native-like can be formed via the sequential formation of the 5-55 and 14-38 disulphide bonds, however these then hold the final 51 and 30 residues at too far a distance for the native to be successfully formed.
77
Observations of the Disulphide formation pathways of BPTI:
- Folding occurs non-randomly. with equilibria biased (kinetically preferred) in the formation of specific intermediates that drive folding.
- 'Randomness' of folding can be increased by increasing the concentration of denaturant (e.g. urea)
- Kinetically preferred non-native disulphide bonds may form to increases the kinetic favourability
- Native-like structures may form in many stages of formation, however these can either favour or inhibit native formation, dependent on the requirements of subsequent rearrangements.
78
BPTI: What occurs to dead-end products?
Either rejoin the pathway through reverse equilibria reactions, or are recycled by protein recycling system.
79
BPTI: Free energy stability
Show the formation of each distinct intermediate state, This also suggests which intermediate species will be present in the absence of disulphide bonds.
80
Why are disulphide bond intermediates easier to characterise?
The intermediates can be made very stable and studied at leisure.
81
What characterises Amyloid disease?
The deposition of protein within tissue.
82
What are the examples of Amyloid Diseases?
Alzheimer's, Cerebral amyloid angiopathy, Prion diseases, Kuru, Parkinson's, Type 2 Diabetes, Rheumatoid arthritis.
83
How may amyloid fibre structure differ?
Amyloid fibres will adopt different packing arrangements, leading to multiple polymorphs for different diseases.
84
What is the general structure of an amyloid fibre?
Structured unfolded arrangement, beta sheets forms at right angles along the axis of the fibre.
85
Why dye technique is used to identify amyloid plaques?
Red Green birefringence.
86
Red Green Birefringence:
Transect of tissue take, congo red is added, the sample is put in a microscope with polarisers, when the polariser below the sample is turned to a right angle of the polariser above, the dye appears green. (due to the regular ordering of the dye upon binding to the lattice)
87
What techniques characterised the general structure of amyloid fibres?
X-ray diffraction and NMR spectroscopy.
88
Example of amyloid fibre structure that's been found and characterised?
The Alzhiemer's associated- ABeta.
89
What are Prions?
Proteinaceous infectious particles, a type of transmissible spongiform encephalopathy (TSE)
90
Examples of Human prion diseases:
Kuru, Creutzfeldt-Jakob disease (CJD - most common prion disease), Fatal Familial Insomnia (FFI)
91
Examples of Animal Prion Diseases:
Scrapie, Chronic wasting disease (CWD), Bovine Spongiform Encephalopathy (BSE - epidemic in the 1990s when cow meat used in cow protein supplements)
92
Characteristics of Prion Diseases:
-Neuronal Cell death
-Transmissible between individuals and generations
-Normal Cellular prion protein (PrPC) -> essential for infection but function unknown.
-Protein only hypothesis (No nucleic acid, bacteria, or viruses)
- Unknown cytotoxic agents
93
What are the key PrPs associated with prion infection?
PrPC - essential but unknown function
PrPres - forms amyloid-like deposits that are resistant to proteases.
94
What's the difference between (infectious) PrPSc (scrapie) and PrPC?
PrPsc can act as a template for its own self assembly, catalysing the conversion of PrPC into PrPSc.
95
Organisation of the PrP:
N-terminal Domain: Mostly unfolded region (intrinsically disordered protein) with an octapeptide repeat region (high affinity for metal ions)
C-terminal Domain: alpha helical GPI anchor (attaches to membrane) attached to a larger structured region. Two glycosylation sites (N181 and N197) joined by a disulphide bond in the folding domain.
96
Cross section of a prion protein:
- Shows the layers of hydrogen bonding between parallel peptide chains, each forms a hair pin structure to allow for species in proximity to form disulphide bonds (between glycosylation sites in folding region).
97
What region of the human prion protein makes up the core?
Residues 160-220. In the presence of a C-terminal 145 stop codon, the disease still occurs.
98
Does PrP have multiple folding conformations?
Yes! PrPC has additional confirmation. PrPC has an unstable fold for the C-domain, capable of folding in stages than 1 clearly cooperative unfolding transition.
99
Significance of Partial unfolding of PrPC?
The partial unfolding events cause accessibility of parts of the protein to become involved in amyloid forming interactions. This is a source of heterogeneity across fibres produced by amyloid protein, and also mean that conclusions based on mutations to the sequence will not reflect the propensity of mutant proteins to become amyloid.
100
PrP fibre formation: What is the first major aggregate structure formed?
Initially lots of granular species form (many people think these to be the cyotoxic agents) -> these assemble into the larger fibres.
101
Evidence that oligomer formation is the disease causing agent rather than larger amyloid:
The folding and assembly of large amyloid assemblies are not required for symptoms to occur.
When the species formed in the pathway leading to amyloid fibres were partitioned according to their sizes, the infective species are the smaller molecules (17-27nm).
102
What is the structure of the infectious prion unit?
Shorter along the long axis of amyloid fibres, but greater lateral association.
103
What are cystatins?
Central molecules in cause of cerebral amyloid angiopathy
104
What are the symptoms of cerebral amyloid angiopathy?
strokes, death
105
How does cerebral amyloid angiopathy damage the brain?
Deposits of A-beta and human cystatin C leads to the destruction of smooth muscle cells around the blood vessels of the brain -> less resistance to blood pressure -> brain bleed.
106
Cystatin structure:
Dimer, N-terminal end (on top with conserved Val 55 and Pro 1102 loops.
And a Pro74 (loop 2)
107
How do cystatins function?
Cystatins are cysteine competitive proteinase inhibitors that inhibit various cathepsins. They insert their Val55 and Pro1102 loops and N-terminal region into the active site of the proteinases, avoiding cleavage through their inappropriate geometry for the catalytic groups.
108
What are the two conformations Cystatin can take?
Trans conformation (more stable) is the folded state, in this state its valine is in a strained conformation, and its backbone at torsional angles not in ranges normally found in folded proteins.
109
Dimerisation of Cystatin:
- Dimerisation rate is very slow, it's a bimolecular reaction (requiring two molecules come together in the rate limiting step) -> It's heavily reliant on the concentration of Guanidinium Chloride (denaturant), requiring a large conformational distortion (3D domain swapping)
-During the process the proteins unfold and refold intertwined with the other monomer.
- Loop 1 'disappears' and so strand 2 doesn't loop complete its loop with its monomer strand 3, instead binding to strand 3 of the other connected monomer.
110
Cystatin Dimer vs monomer?
Dimer: Inactive, more stable, lacking strain from valine (loop 1 removed)
Monomer: Active, not as stable -> more likely to unfold
111
Tetramerisation of cystatins:
- Unimolecular reaction requiring the isomerisation of Pro74 of loop II.
- Very slow rate
- Tetramer formation requires less folding
- Conformational distortion required (molten globule) -> form hydrophobic core
- The isomerised dimers join together by their H75 residues.
112
Multimerisation of Cystatins:
Making a free energy diagram of the each process using their guanidinium rate. Energetically the the process of tetramerisation and multimerisation are indistinguishable. Hence the the formation of multimers to form amyloid fibres is dependent on the isomerisation of the same loop II proline.
113
Importance of proline isomerisation: cystatins
Proline isomerisation is associated with the self-assembly of beta-sheets, many of these native protein states have their residues at the end of these sheets as 'beta bulges' -> these proline residues control the presence of these, to stop larger molecules that tend towards becoming monomeric, from assembling into the veery large ordered structures (found in amyloids)
