L5 - Early stages in protein folding

Question 1

What are the 2 stages of protein folding?

Answer 1

Can either fold directly into the folded state or has later folding

Early folding is very rapid and the later stage is slower

Chaperones are stopping off pathway interactions

Question 2

How do we measure protein folding?

Answer 2

Protein folding in vivo (in the cell) is difficult to study

So most of our data comes from experiments on protein folding in vitro (in the test tube)

We assume that the basic rules are the same

So we believe that in vitro studies can tell us the rules of protein folding

Question 3

How are in vitro protein folding experiments done?

Answer 3

Purify proteins from a natural source

Denature proteins by using denaturants which bind to peptide backbone & inhibit the formation of secondary structure

Then dilute the solution & allow the proteins to ‘refold’

Question 4

What denaturants are used in in vitro protein folding experiments?

Answer 4

Either urea or guanidine – they look a bit like a peptide bond

They directly compete with the H bonds in the backbone

Need to use high concentrations of these

Solvent denaturation

Question 5

What are the different types of in vitro denaturation methods?

Answer 5

Solvent denaturation

Thermal denaturation

Acid or base induced denaturation

High pressure denaturation

Question 6

Solvent denaturation

Answer 6

Urea, guanidine-HCl – most popular method in refolding studies

Question 7

Thermal denaturation

Answer 7

Often irreversible so not much use in refolding studies – eg. scrambled egg

Question 8

Acid or base induced denaturation

Answer 8

If pKa’s of side chains are different in folded & unfolded states folding becomes pH sensitive

Example: Glu

• pKa(unfolded) = 4.5
• pKa(folded) = 1.5
• At pH 7 Glu is ionised but in both states at pH 4.5, 50% of Glu in unfolded stated is protonated
• In folded form highly ionised so folding is faster at high pH

Question 9

High pressure denaturation

Answer 9

At high pressure water is pumped into the spaces in the hydrophobic core overcoming the hydrophobic effect & the protein denatures

Question 10

From the unfolded to the native

Answer 10

The unfolded state – theoretical calculations suggest that it is a population of a wide range of conformers, not only extended (cooked spaghetti) but also many different compact ones

The folded state – in contrast is highly compact & ordered with many specific interactions

By diluting the urea, most proteins will go from unfolded to folded

Question 11

The folding reaction

Answer 11

Can have lots of different unfolded states

There are quite a lot of intermediates and those intermediates can adopt different forms so it can be incredibly complicated
It is possible to have multiple unfolded states trying to converge

Question 12

Chaperones in protein folding

Answer 12

No not tell proteins how to fold

Alter the energy level of intermediate stages to block a lot of off-pathway forms which might aggregate in the concentrated environment of the cell

Increase the speed of folding down the most direct pathway – funnel protein folding down the fastest route

With the high concentration of proteins in the cell, it is important to have chaperones

Question 13

What are the elementary steps in going from an unfolded (U) to a folded (N) protein?

Answer 13

• Secondary structure formation
• Hydrophobic contacts – core formation
• Close packing of side chains etc.
• Specific tertiary interactions (disulphide bridges)

So if we can track these events, we can tell whether & when & to what extent the protein is folded

Question 14

What do the energy barriers in free energy diagrams indicate?

Answer 14

Indicates that there are going to be intermediates (I)

I exists as a recognisable step between D and N

The deeper the dip, the longer and more stable the intermediate will be

Some proteins have well defined intermediates and some do not
Intermediates often look quite close to the native state

Question 15

3 techniques to measure protein folding

Answer 15

Tryptophan fluorescence

Circular dichroism (CD)

Nuclear Magnetic Resonance Spectroscopy (NMR)

Question 16

Tryptophan fluorescence in measuring protein folding

Answer 16

Fluorescence occurs when a molecule that is excited with light at a particular wavelength emits light at a longer wavelength

In proteins tryptophan residues absorb at 280 nm & emit at either 330 nm (folded) or 350 nm (unfolded)

Tryptophan is a highly fluorescent naturally occurring side chain

Question 17

Circular dichroism (CD) in measuring protein folding

Answer 17

Is a method which uses polarised light to measure the quantity of secondary structure in a protein

Secondary structure absorbs polarised light because it is composed of ordered L-amino acids

Question 18

Nuclear Magnetic Resonance Spectroscopy (NMR) in measuring protein folding

Answer 18

Based on the magnetic spin states of atomic nuclei & the interactions between them

In 1 form the technique works only for atoms if it contains an off number of neutrons + protons

It can detect 1H hydrogen isotope (H, most abundant) but not 2H (deuterium, D)

Question 19

What is fluorescence?

Answer 19

A process whereby photons absorbed at 1 wavelength cause the emission of photons at a longer wavelength

Can selectively pick out certain things in images

Question 20

Fluorescence & protein folding

Answer 20

The unfolded is at a longer wavelength & a lower intensity
The folded state is at a lower wavelength & a higher intensity

Water is removed as the protein folds

Photons coming off the protein tell you whether its from a dry (folded) or wet (unfolded) environment

Question 21

How do we measure fluorescence?

Answer 21

We only have to illuminate the proteins with light

Done in a spectrometer

Exciting protein in a cuvette & observe the wavelength

It shifts when the protein folds

Question 22

Why does the fluorescence shift?

Answer 22

When in hydrophobic environment, tryptophan emits light at 320-330 nm

When in hydrophilic environment, tryptophan emits light at 345-355 nm

Thus it can be used as a measure of protein folding since Trp (W) is usually in the core of a folded protein

Because the tryptophan is normally in the core of the folded proteins, as the tryptophan is suppressed inside the structure of the folded protein, the fluorescence shifts

This is telling us about the formation of the hydrophobic core of the protein

Question 23

Circular dichroism spectroscopy

Answer 23

Proteins contain all L-amino acids

Since peptide bonds absorb light & are part of the protein stereoisomer they are also optically active

Depending on how the peptide backbone is folded this can rotate the plane of polarised light left or right – get wavelength dependent rotation

If we measure this rotation we can measure the amount & type of secondary structure

Question 24

What is CD spectroscopy measuring?

Answer 24

The formation of secondary structure (alpha helices and beta sheets) and these tend to get formed in early protein folding

All proteins contain L-amino acids so when they are illuminated with light, because they are single optimal isomers, they have the ability to rotate the plane of polarised light

At particular wavelengths the L-amino acids have the ability to rotate the plane slightly and this is dependent on the secondary structures

Question 25

Results from CD spectroscopy

Answer 25

When the spectrophotometer goes from a negative signal to a positive signal, this is the protein going from unfolded to folded, as the secondary structures are formed

This experiment is also very easy to conduct compared to other techniques, such as NMR, as it simply involves shining light to the protein and the protein does not need to be chemically modified in any way

The rate at which the wavelength changes, can tell us the rate at which a particular secondary structure is formed

Question 26

Why is the very early stage of protein folding not easy to see?

Answer 26

Folding of proteins happens on a very short timescale

In most cases, using conventional spectroscopic techniques we just see the end products (N or U)

Question 27

How can we measure the very early stages of protein folding?

Answer 27

Rapid mixing techniques allow us to measure protein denaturation for folding processes in real time (microseconds or smaller time scale)

Can denature a protein & follow its renaturation right from the ‘beginning’ & measure the rates of the elementary steps of protein folding using such mixing chambers within fluorescent or CD spectrometers

Question 28

What is stopped flow?

Answer 28

Stopped-flow is a lab technique for studying fast chemical reactions

Question 29

Process of stopped flow experiments

Answer 29

A = denatured protein in urea
B = buffer to dilute the denaturant
A & B are mixed at chosen ratios

Stop syringe stops flow & spectroscopy observes the evolving reaction
Then it is in stop flow conditions

The proteins then fold in the cuvette
Important that it takes only a few msec for mixture to arrive in the optical path

The dead time, which is a couple of ms, it is possible to follow folding from dead time to several hours as it has been stopped

Question 30

What is dead time in stopped flow experiments?

Answer 30

As found in many proteins, there is an initial burst phase, during which time some of the native-like fluorescence and a substantial fraction of the native content of secondary structure is formed

The intermediate formed within mixing time

Question 31

Results of fast folding experiments

Answer 31

These methods show that some proteins form short parts of helix very early (us)

Much of the early folding forms secondary structure – eg. H bonds, but incomplete tertiary structure

In either case this is very fast; how does it avoid Levinthal’s paradox?

Question 32

Models of early protein folding

Answer 32

2 main models:
• Framework model
• Hydrophobic collapse model

Proteins have to be guided down pathways, it is not random, in order to fold as quickly as possible

Early folding is probably a mixture of these 2 processes & leads to the next stage of folding

Question 33

Framework model for early protein folding

Answer 33

Some short stretches from helices very fast – residues with high propensity for helix formation (A, E, M, L)

Energy gained from H bond formation

The forms a framework which guides the rest of the protein to fold – like a zip fastener

Beta structure can acts as a framework but is slower to form so more likely to go wrong – might be the origin of prion disease & Alzheimer’s

Question 34

Hydrophobic collapse model for early protein folding

Answer 34

Rapid exclusion of water gives a big energy input from the hydrophobic effect

Protein is thus compact & the early secondary structure interactions are guided by the reduced number of structures present

This model says that in achieving dehydration of those hydrophobic side chains it brings the core of the molecule together and this then drives the rest of protein folding