2 - Protein Folding Dynamics

Question 1

How is protein folding involved in proteostasis?

Answer 1

Regulation of protein folding is one of the primary methods of proteostatic control. This can take the form of controlling the ratio of correctly folded to incorrectly folded proteins, and hence the number of functional proteins and how many are allowed to immediately misfold and be targeted for degradation.

Question 2

Other than in relation to disease, what is the main purpose of understanding protein folding?

Answer 2

To be able to predict it from sequence. Not only would this in fact inform what we know about misfolding diseases, but it may lead to the design of novel folds and features within them that lead to new catalytic functions for use in pharmaceutical and industrial technology.

Question 3

Why is protein folding difficult to predict from sequence?

Answer 3

it involves estimating the interaction between every atom of the protein and the strength of this relative to the bonds made with the aqueous environment. Such parameters are difficult to measure with precision.

Question 4

What occurs to the nascent polypeptide chain?

Answer 4

Proteins can either be folded where they are translated, or transported to another part of the cell for this. Those not translated in the cytoplasm are often co-translationally deposited into the ER where it may obtain some post translational modifications and/or structure

Question 5

What happens to proteins targeted to the Golgi Apparatus?

Answer 5

Further post-translational modification (eg proteoglycans) and sorting.

Proteins leaving here may be targeted to the late or early endosome (and on to the lysosome) or by secreted from the cell directly (eg via the bacterial SecB-A system) or in secretory vesicles. Some proteins are only folded after they excreted.

Question 6

What happens to proteins translated straight into the cytosol?

Answer 6

Proteins that are deposited straight into the cytosol are often transported to the nucleus, mitochondria, peroxisome or other plastid to be folded there.

This process is controlled by sorting signal amino acid codes that are often cleaved off upon reaching the destination. Structures such as the mitochondria possess their own chaperones for folding proteins there.

Question 7

What defines protein stability?

Answer 7

the difference in free energy between its native and unfolded states.

Question 8

What is the typical range of protein stabilities, and what does this indicate?

Answer 8

For most proteins this is around 5-15kcalmol-1. This is very low – the difference in stability between the native and unfolded states often corresponds to only a few H-bonds. Hence there is a fine balance/equilibrium between the folded and unfolded states.

Question 9

What defines the folded state of a protein?

Answer 9

The folded state is defined as the ‘lowest free energy state’ of a protein.

Question 10

What determines the change in free energy of a protein?

Answer 10

The combination of the changes in enthalpy and conformational entropy. For the lowest ΔG a very negative change in enthalpy or a large increase in conformational entropy is required.

Question 11

How much does the enthalpic change of folding contribute to the protein stability?

Answer 11

The enthalpic value of the various intra-protein interactions that make up the structure account for little in terms of the free energy change.

Question 12

What do the various intra-protein interactions contribute to the enthalpic change in folding?

Answer 12

• Internal H-Bonds – 2-5 kcalmol-1
• Charge-charge interactions – 5 kcalmol-1
• Van der Waals interactions – 0.01-0.2 kcalmol-1 per atom pair

Question 13

Why do enthalpic considerations have little effect on the free energy change of folding?

Answer 13

Many of the favourable interactions that the protein can form can aso be made by interaction with water, hence ΔH contributes little to the ΔG – protein folding is a largely entropic effect.

Question 14

What drives protein folding by decreasing the free energy of the folded state?

Answer 14

The increase in entropy that comes from the hydrophobic effect, though some of this is negated simply by the lower entropy associated with having any kind of stable conformation.

Question 15

What is Levinthal’s Paradox?

Answer 15

Cyrus Levinthal’s 1968 paradox describes the discrepancy between the time it takes a protein to fold (less than one second) and the time it would take a protein to attain the correct conformation by random sampling. With 10^50 different conformations and a rate of one conformation per 10-13 seconds, it would take 10^30 years to sample them all.

Question 16

If I met somebody called Cyrus Levinthal, how fast would I have their babies?

Answer 16

Faster than is thought to be biologically possible. All of my children would take the name of their father and we would rejoice.

Question 17

What flawed assumption does Levinthal’s paradox make? What is the truth of the matter?

Answer 17

That the sampling is unbiased. Levinthal imagined that each side chain could rotate around its single bonds at random, with no stabilisation for any of them until all were in the correct conformation.

In reality conformational bias guides the formation of the correct structure through the formation of intermediates – and any move towards a more stable structure is stabilised often regardless of whether or not the rest of the protein is in the ‘correct’ conformation.

Question 18

What are the three classical models of protein folding?

Answer 18

The Framework Model
The Hydrophobic Collapse Model
The Nucleation-Condensation Model

Question 19

What is the framework model?

Answer 19

The Framework model suggests that secondary structural elements form independently, which induces coalescence of the tertiary structure.

Question 20

What is the Hydrophobic Collapse Model?

Answer 20

The Hydrophobic Collapse Model suggests the oppostie of this, with the initial formation of a hydrophobic core that allows for the secondary and teritary structure to form around it.

Question 21

What is the Nucleation-Condensation Model?

Answer 21

The Nucleation-Condensation Model acts as a compromise between the two others, describing the formation of a nucleus of secondary and tertiary structure which acts as a template for the folding of the rest of the protein through hierarchical assembly.

Question 22

What is a protein folding landscape?

Answer 22

Also called folding funnels, these are a method of plotting the different ways in which a protein might fold by measuring the decrease in conformational entropy as a function of the decrease in enthalpy.

Question 23

How do protein folding landscapes indicate a biased directionality to protein folding?

Answer 23

The folding funnel shows that the unfolded state (D) is highly heterogenous, so there are a number of routes to reach the folded state (N). As the (more stable) native contacts form, the number of available conformations decreases, thus driving folding in the right direction

Question 24

What can analysis of the thermodynamic states seen in a folding funnel show?

Answer 24

They can elucidate the formation of folding intermediates, partially folded states, oligomers, amorphous aggregates and amyloid fibrils.

Question 25

What folding funnel does an IDP produce?

Answer 25

Lack of any significant troughs.

Question 26

What does the defining of a protein folding pathway entail?

Answer 26

Determination of all the structures of the N (native) I (intermediate) and U (unfolded) states. Cellular conditions are difficult to work with or to replicate, so this study is typically done in vitro.

Question 27

How are folding intermediates characterised biochemically?

Answer 27

Equilibrium and kinetic studies are often used as a wet-lab experimental approach, often in conjunction with computational and molecular modelling approaches.

Question 28

How is protein folding directly studied?

Answer 28

To produce data on the folding or unfolding of a protein, an experiment must be devised in which a change in signal is produced between the folded and unfolded states.

This often uses denaturants or changes in conditions such as pH and temperature to disrupt protein structure, using this as a model for the reverse process.

Question 29

What techniques are used to measure the changing protein structure during folding?

Answer 29

• Intrinsic fluoresence
o Analysis through changing environments of Trp residues
• ANS Binding
o Analysis of exposure of hydrophobic surfaces
• Circular Dichroism
o Changes in the secondary/tertiary structure
• Hydrogen Exchange/NMR
o Formation of H bonds
• Atomic Force Microscopy/Optical Tweezers
o Forces within a protein chain can be analysed by extension

Question 30

How quickly does protein folding occur?

Answer 30

Protein folding is a rapid process, occurring in the range of microseconds to seconds, so the intermediate states are generally only transiently populated

Question 31

What can equilibrium studies show about a protein folding pathway?

Answer 31

Equilibrium studies look at the fraction that is folded/unfolded as a function of the concentration of denaturant. This can show the protein’s stability, as well as characterising highly populated intermediates.

Question 32

What can kinetic studies show about a protein folding pathway?

Answer 32

They look at the change in the fraction that is folded/unfolded as a function of time, to determine the number of steps in the process and its rate. This can allow for detection of intermediates.

Question 33

How are kinetic studies carried out?

Answer 33

This often uses stopped flow apparatus to rapidly mix the folded protein with a denaturant, or by refolding the protein by mixing the unfolded solution with one lacking denaturant. This allows the changes in structure to be measure by CD or fluorescence on a millisecond to second timescale.

Question 34

What is a Chevron Plot?

Answer 34

The plotting of the kinetic analysis of both folding and unfolding on the same graph, measuring foldedness against denaturant concentration.

Question 35

How is intrinsic fluorescence used in folding analysis?

Answer 35

Although tyrosine and phenylalanine are also naturally fluorescent, tryptophan residues are predominantly used as they give the highest quantum yield.

The environment around Trp affects its emission intensity and hence wavelength, so Trp can act as a reporter for changes in the protein structure. The more solvent exposed the Trp is the longer the wavelength.

Question 36

How is Circular Dichroism used in folding analysis?

Answer 36

By using far-UV circularly polarised light – which is rotated differently by helices and beta sheets – the secondary structural changes of a protein can be measured dynamically.

Analysis of the change in the signal during folding or unfolding can map the large conformational changes.

Question 37

How can NMR be used to measure the foldedness of a protein and hence track folding?

Answer 37

When analysing protein folding, a HSQC (heteronuclear single quantum correlation) spectrum is often used which uses 15N labelled proteins to produce a graph showing the different environments of every N-H pair.

This produces a fingerprint of the protein, and the changes in these peaks (in terms of chemical shift or broadening) can allow for the examination of the movement of each individual residue

Question 38

How is Hydrogen-Deuterium Exchange NMR used to observe protein folding?

Answer 38

H2O moieties in a macromolecule can be exchanged for D2O, in a process dependent on the level of solvent exposure. This is useful as it allows estimation of the solvent exposure of different residues, especially when trapped states are examined by NMR or by Mass-Spec.

Question 39

How is protein engineering used to analyse folding?

Answer 39

This is a technique pioneered by Fersht in 1989, allowing examination if the folding pathway in detail using the Phi Analysis Principle.

To do this, they use a variety of mutant proteins in which specific side chain interactions known to occur are inhibited. This allows the effect of the sidechains on folding to be assessed via analysis of the thermodynamic properties, particularly the ΔG of the mutant and wild type folding events.

Question 40

What is a two-state protein folding pathway?

Answer 40

Only the native and unfolded states are present - no intermediates.

Question 41

What is a multi-state protein folding pathway?

Answer 41

Multistate folding pathways possess an intermediate, usually a single one leading to a three-state folding pathway.

Question 42

What form might protein folding intermediates take?

Answer 42

These may be a molten globule within which the chains rearrange to produce folded regions, or the intermediate may be the formation of the first folding domain before the rest of the protein folds.

Question 43

What is Alpha-1 Antitrypsin?

Answer 43

This is a serpin protein with many different folding conformations, whose role is to inhibit elastase in order to protect the lungs.

The protein can fold into its normal active state, or into an alternate one which is able to polymerise into inactive chains.

Question 44

What does Circular Dichroism study of the unfolding of Alpha-1 Antitrypsin show?

Answer 44

It shows no intermediate state, as the decrease in the ellipticity signal follows the two-state pattern.

Question 45

What does fluorescence study of the unfolding of Alpha-1 Antitrypsin show?

Answer 45

The fluorescence analysis of the five Trp residues show them becoming more exposed, the maximum wavelength shifting from 338nm to 355nm.

Question 46

What do these studies suggest about Alpha-1 Antitrypsin misfolding?

Answer 46

That the misfolded intermediate is normal transient intermediate in the folding pathway.

Question 47

What is the structure of lysozyme?

Answer 47

This is a small (129 AA) monomeric protein with two domains; an alpha domain containing four helices and a second domain largely comprising a triple-stranded beta sheet.

Question 48

What did CD, fluorescence and ANS binding assays show about the lysozyme folding pathway?

Answer 48

CD and fluorescence studies showed rapid (

Question 49

What did H/D exchange analysis by Mass-Spec show about the lysozyme folding pathway?

Answer 49

That the two domains fold at different rates, with the alpha domain being the quicker. This did find intermediate states in the folding pathway in which the second domain was not yet properly folded.

Question 50

What is the lysozyme folding pathway?

Answer 50

It begins with a rapid collapse during which some helical secondary structure begins to form.

This is followed by the formation of some tertiary structure resembling the alpha and then beta domains that now resemble the native state, but lack critical interactions.

Then the secondary structures coalesce in the much slower step, mutually stabilising each other and properly positioning their side chains.

Question 51

What are the difficulties involved in designing novel proteins?

Answer 51

Creating a new protein from scratch involves not only creating a viable 3D structure, but also putting it in a primary structure that will fold into that conformation naturally.

Question 52

What is an example of a artificially designed protein?

Answer 52

Kuhlman et al in 2003 described the design of a protein they named Top7 using the Rosetta program to theoretically verify the in silico models. This small protein was produced and analysed extensively using an array of biophysical techniques as well as NMR

Question 53

What did analysis of the designed Top7 protein show?

Answer 53

Crystallography using seleniomethionine produced a resolution of 2.5Å, enough to show that a novel topology had been created, with a very high similarity to the predicted structure.

Hence this showed that protein folding can be predicted for very small polypeptides.

Question 54

What does protein design such as Top7 not account for?

Answer 54

In vivo translation.

This is a far more complicated affair, as it required the complex conditions into which the protein is being translated to be taken into account.

It also requires understanding of the differences between in vitro folding and folding while emerging from a ribosome.

Question 55

What complications does in vivo folding present?

Answer 55

Complications such as nascent chain folding, varying translational speed, interactions (eg electrostatics) with the ribosome itself, action of chaperones, the dynamic implications of the tethering and the influence of surrounding sequence.

Question 56

What effect does nascent chain folding have on the protein folding landscape?

Answer 56

Nascent chain folding is thought to affect the folding in such a way that it allows for tunnelling between intermediates and the native state on the folding funnel.