Proteases and AFM Flashcards
Describe proteases?
Proteolytic cleavage of peptide bonds is one of the most frequent and important enzymatic modifications of proteins
Four main classes of proteinases: serine, cysteine, aspartic acid and metallo-
Metallo have a metal ion in the middle of the proteolytic site
Many proteases are relatively unspecific towards their substrate:
trypsin cleaves C-terminal to positively charged side-chains
pepsin cleaves C-terminal to hydrophobic side-chains
Many ‘passive’ proteases function in an extracellular environment:
Gastric enzymes, blood clotting proteases, protein processing and lysozyme
Other ‘passive’ proteases are excluded from the cytoplasm by sub-cellular compartments
Lysosomal enzymes and bacterial periplasmic proteases e.g. DegP
Describe the structure of protein to be degraded?
In order for proteolysis to occur the bonds susceptible to the protease must be accessible
Cleavage commonly occurs in dynamic regions such as domain junctions and loops
However, fully folded native domains can be highly resistant to degradation
What experiment can we do to see the exposure of a protein a degradation?
Enzyme is unfolded with denaturant, refolded over time and then we use an activity assay to see the protein gradually refold
If we do a trypsin assay - the enzyme has exposed and can be completely degraded - but it was left for a time, it is will completely refold and therefore is almost resistant to proteases
What problems do we have with proteases?
How are proteases regulated in the cytoplasm?
How can very stable proteins be degraded?
How are proteases regulated in the cytoplasm?
The proteolytic active sites are enclosed within a chamber, only proteins that can gain entry will be degraded
Selectivity is achieved by making the opening very narrow (1-2 nm diameter) pores
This is smaller than the dimensions of most folded proteins
How can very stable proteins be degraded?
Use energy to unfold stable proteins faster than intrinsic rate
Proteases starts to pull the protein through the hole
This takes place in many domains of life
In chambered protease degradation, proteins are actively unfolded by ATP driven motors
Linking unfolding to an energy dependent process allows chemical energy to be harnessed as mechanical force, degrading very stable proteins
Ubiquitinated molecules are brought to the proteasome - but this is a very complicated chambered protease
Describe bacterial chambered proteases?
There are five chambered proteases in bacteria: ClpXP, ClpAP, HslUV, FtsH and Lon
In most these the protease and the unfoldase (an ATPase from the AAA+ protein family) are present on different polypeptide chains.
FtsH and Lon both activities are present on a single polypeptide chain.
Most work performed on ClpXP and ClpAP
Describe the bacterial Clp (caseinolytic protease) proteases?
Clp P: 2 stacked rings of heptamers
Each monomer contains a serine protease active sites
Clp P can bind to two different AAA+ proteins:
Clp A: a ring like hexamer can bind at each end of Clp P
This protein has two AAA+ domains
Clp X: a ring like hexamer can bind at each end of Clp P
This protein has one AAA+ domain
The substrate is tagged - the tag is engaged - it starts to unfold by pulling through the hole and then degrading/translocation takes place
Describe the AAA+ superfamily?
ATPases Associated with various cellular Activities
200-250 amino acid ATP-binding domain
AAA+ proteins usually form oligomeric ring-like structures (hexamers)
These hexamers participate in a wide range of cellular activities either independently or with other proteins: Protein degradation Protein disaggregation (ClpB) - forms reactivated smaller folded proteins Protein-complex remodelling - tugs on a SNAP-SNARE complex and breaks it up ClpX can remodel if not complexed with ClpP
How does ClpXP recognise the substrate?
Clp X binds to specific sequences or ‘tags’ present at the N or C-termini (or even internally) of the substrate protein
These tags label specific proteins for degradation
The most intensively studied is the ssrA tag added to polypeptides on stalled ribosomes
tmRNA lands on the ribosome and sticks a sequence on the protein to degrade it - with the specific sequence being AANDENYALAA
The AANDENYALAA sequence has two binding motifs:
1. LAA is recognised by loop regions (GYVG) in the pore of the ClpX hexamer
2. AANDENY is recognised by an adaptor protein called SspB
This also binds to the substrate protein and ClpX, increasing the affinity of the substrate for ClpX
As many cycles of ATP hydrolysis sometimes have to be applied to degrade a single protein, this adaptor protein prevents dissociation of the substrate ClpXP complex
How does the AAA+ unfold the substrate?
By looking at using different denaturants we can see relatively slow degradation of the proteins
However, using no denaturants but using Clp AP - we find the protein denatures very fast
Therefore:
The AAA+ domain utilises ATP to catalyse the unfolding of substrate proteins
GFP behaves as if it were dissolved in denaturant
Clear evidence the AAA+ is essential in speeding up the unfolding of proteins
What is force localisation in the proteasome?
As force acts locally, the rate at proteins are degraded should reflect the stability (or dynamism) of regions of the substrate near to the point of tag location
If the protein is mechanically weak near the tag - increases the rate at which the proteins are unfolded
Increase mechanical stability = decrease the rate of degradation
The number of ATPs hydrolysed to degrade a single substrate protein increases with increasing mechanical stability
What are single molecule manipulation methods?
Atomic force microscopy (cantilevers) and laser traps / tweezers are the most common methods to measure the forces applied onto/by biomolecules
Despite using different apparatus both measure the displacement of a ‘spring’ of known stiffness to measure the force applied
AFM good for measuring mechanical properties over short distances. Laser tweezers better for small forces over longer distances
What is the AFM?
Atomic force microscope:
It is used as a force sensor
We pull on the tip, which moves the laser up/down - which is detected by the photodiode
The spring constant of the cantilever can be measured
This allows the force applied by/onto a bio-molecule to be quantified
AFM measures pN-nN forces (>25 pN)
The height of surface features can be measured because the relationship between the distance the tip has moved and the change in voltage of the photodiode can be quantified
For this application the sample is moved under the tip and the image is built up line by line
What are the two modes of AFM?
Contact mode
Tapping mode
Describe contact mode of AFM?
The tip is moved along the surface and the height is moved up or down to maintain a pre-set contact force
Advantages: easy to use, any type of tip can be used and can be varied to optimise contrast
Disadvantages: sample damage and high lateral forces can displace bio-molecules from the surface
Describe tapping mode of AFM?
A high frequency high amplitude sinusoidal oscillation is applied to the tip
The cantilever is moved up or down to maintain the amplitude of this oscillation
Advantages: less sample damage/distortion
Reduced lateral force
The difference in phase between the driving oscillation and tip oscillation gives additional information
Disadvantages: specific tips required, more difficult to implement
Describe the cantilevers and the resolution in AFM?
The sharpness or ‘aspect ratio’ of an AFM cantilever is a key determinant of resolution
Using a ‘blunt’ (low aspect ratio) tip will both broaden the image and under-estimate the depth of some features
Sharp tip = high resolution images
You can add carbon nanotube onto the tip for a very sharp tip - but very hard to achieve
Describe resolution in AFM?
- Under ideal conditions (a very high vacuum, on a flat sample adhered to an atomically flat surface) and using a cantilever derivatised with CO (good contrast) it is possible to resolve individual bonds within a molecule e.g. pentacene
- For biological samples which are generally globular and softer, the best images are obtained from 2D crystals
This can give a resolution of 1 nm - For non-crystalline biological samples, the achievable resolution varies markedly
The resolution depends on whether the image is taken in solution, how the molecule is immobilised and the properties of the material
Resolution is 5nm
What are some applications of AFM that links to biochemistry?
Low resolution structure in absence of other data
Amyloid formation
There is a lag phase where nuclei grow - at each of these points along a graph we can take AFM pictures
There is a rapid increase where we can see a large amount of protein structure in solution at that moment
Measurement of individual populations in a mixture
Amyloid seeds were grown so the fibral core had an epitope
They added an antibody to the epitope to identify where the fibral seed was - to monitor the growth of the seed
They saw the fibres grow from one end and some grow more than others
