L5 - Early stages in protein folding Flashcards
What are the 2 stages of protein folding?
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
How do we measure protein folding?
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
How are in vitro protein folding experiments done?
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’
What denaturants are used in in vitro protein folding experiments?
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
What are the different types of in vitro denaturation methods?
Solvent denaturation
Thermal denaturation
Acid or base induced denaturation
High pressure denaturation
Solvent denaturation
Urea, guanidine-HCl – most popular method in refolding studies
Thermal denaturation
Often irreversible so not much use in refolding studies – eg. scrambled egg
Acid or base induced denaturation
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
High pressure denaturation
At high pressure water is pumped into the spaces in the hydrophobic core overcoming the hydrophobic effect & the protein denatures
From the unfolded to the native
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
The folding reaction
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
Chaperones in protein folding
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
What are the elementary steps in going from an unfolded (U) to a folded (N) protein?
- 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
What do the energy barriers in free energy diagrams indicate?
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
3 techniques to measure protein folding
Tryptophan fluorescence
Circular dichroism (CD)
Nuclear Magnetic Resonance Spectroscopy (NMR)
Tryptophan fluorescence in measuring protein folding
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
Circular dichroism (CD) in measuring protein folding
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
Nuclear Magnetic Resonance Spectroscopy (NMR) in measuring protein folding
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)
What is fluorescence?
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
Fluorescence & protein folding
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
How do we measure fluorescence?
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
Why does the fluorescence shift?
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
Circular dichroism spectroscopy
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
What is CD spectroscopy measuring?
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
Results from CD spectroscopy
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
Why is the very early stage of protein folding not easy to see?
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)
How can we measure the very early stages of protein folding?
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
What is stopped flow?
Stopped-flow is a lab technique for studying fast chemical reactions
Process of stopped flow experiments
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
What is dead time in stopped flow experiments?
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
Results of fast folding experiments
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?
Models of early protein folding
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
Framework model for early protein folding
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
Hydrophobic collapse model for early protein folding
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
