Proteins (IMcN) Flashcards
What are the 4 basic steps of purifying a protein?
- Grow/obtain cells
- Lyse cells to release proteins
- Centrifuge to remove cell debris
- Purify using fractionation/chromatography methods
What are the 3 differential precipitation (fractionation) methods of purifying a protein and how do they work?
- Salt fractionation = high salt concentration (ionic strength) reduces the solubility of the target protein in the solution, which precipitates it out of solution
- Isoelectric precipitation (pH) = When the pH of a solution is at the target protein’s isoelectric point (pI), the overall net charge of the protein will be zero, and so the protein will precipitate out of solution
- Heat precipitation = Increase the temp of the solution, and have the target protein be heat resistant. All other heat labile proteins will precipitate out of solution, and the heat resistant target protein will be left in solution.
What are the 3 different types of chromatography used for protein purification and describe each of them
Size exclusion/gel-permeation = Separates proteins by size, smaller proteins get trapped in the pores of the beads in the column which slows them down. Larger proteins don’t get trapped in the pores and so filter through quicker than the smaller proteins.
Ion-exchange = Separates by charge. If beads in column are -vely charged, then +vely charged proteins will stick to the beads. The -vely charged proteins will pass through the column. (works vis versa for +vely charged beads). An increase in salt conc. or change in the buffer pH allows the +vely charged proteins to be removed from the beads so that they can be collected as well.
Affinity = Target protein will bind to the antibody in the column that it is specific to, all other proteins will pass through the column. To remove the target protein and collect it, a ligand with a higher affinity to the bound protein is used to detach the protein from the antibody
What is the general principle of gel electrophoresis, and how does SDS-PAGE work, including the 2 chemicals it involves and what they do?
Gel electrophoresis = proteins inserted into wells of a charged gel using micropipettes, and are then separated as they move down the gel, usually by size and smaller proteins travel faster down the gel than bigger proteins
SDS-PAGE = proteins are all denatured and given a negative charge, so are separated by size only
SDS detergent disrupts the tertiary structure of the protein, causing it to unfold and denature. It also gives all of the proteins a negative charge.
DTT is a reducing agent which reduces cysteines (adds on Hs), disrupting disulphide bonds which helps to denature the protein
What is the isoelectric point of a protein, and what happens when the pH is less than, greater than or equal to this point?
pI = pH at which the overall charge of the protein is 0
pH < pI - Protein is +vely charged
pH = pI - Protein has no charge
pH > pI - Protein is -vely charged
What is 2D electrophoresis/Isoelectric focusing?
Where the proteins are separated by charge first, before being separated by size by SDS-PAGE.
A pH gradient and a current is applied to the gel, and the proteins move to a point in the gel where the pH = their pI, as they want to be neutral. (This is done side ways)
The proteins are then separated by size (down the way) using SDS-PAGE, as before.
What is Native-PAGE electrophoresis, and how does this allow us to figure out the quaternary structure of a protein?
Protein keeps it’s shape (isn’t denatured) and charge, so the proteins are separated by shape, size, and charge
Can figure out quaternary structure by comparing a single band on SDS-PAGE to the native-PAGE band, e.g. always monomer at SDS-PAGE (as denatured), so if 292kDa band at SDS-PAGE, and 876kDa band in native-PAGE, then this is 3x greater, so is a trimer
How does Mass spectrometry work?
Protein sample is converted into a gas + ionized. The ionized protein sample is then passed through a magnetic field, where the ions are separated by mass - lightest ions travel the fastest through the charged matrix and reach the detector first.
The protein sample is denatured when it is ionized.
What is ultracentrifugation? What can it be used to figure out about the protein?
The protein sample is centrifuged, and the dense components forms a solid pellet at the bottom of the test tube, while the less dense liquid (target protein) is called the supernatant.
Can be used to figure out the mass and shape of the protein.
What is light scattering?
Light is directed at a purified protein sample, which will scatter the light once it has passed through the sample.
Light scattered from different parts of the protein will have different light intensities.
Theory = Intensity of scattered light = protein conc. x Molecular mass
How do we measure the protein concentration of a sample?
Use protein absorption spectroscopy, by placing the sample in a cuvette and using a spectrometer to measure the absorbance. Use the beer lambert equation: A=εcl to calculate the protein concentration (c)
What 2 methods are used to determine a conformational change in a protein (just names)?
Circular Dichroism
Fluorescence spectroscopy
Describe the Circular Dichroism technique and what it is used for (2)
Measuring the absorbance of circularly polarised light to study actively chiral proteins (left handed and right handed)
On graph = εLH - εRH
Can give a measure of the percentage of different types of secondary structures in a protein
Can detect conformational changes, as can detect changes in the secondary structure when ligands bind
What is fluorescence spectroscopy and what is it used for?
Measures the absorbance and emission fluorescence of a protein sample (2 peaks on graph).
Can be used to detect conformational changes - the fluorescence intensity measured may increase/decrease when a ligand binds to it
Which techniques are used to determine the quaternary structure of a protein and what does each technique tell us about the protein? (6)
SDS-PAGE + Mass spectroscopy = molecular mass of the protein when it’s denatured (mass of individual subunits)
Size exclusion/gel-permeation chromatography + ultracentrifugation + light scattering = size (mass) and shape of protein, when it ISN’T denatured (can compare this to mass of individual subunits to figure out the number of subunits in the protein (the quaternary structure)
2D electrophoresis = same as native-gel
How are 2D gels used to show the proteome?
Isoelectric focusing and SDS-PAGE is used. Protein spots can then be visualised, with each spot corresponding to a different protein (can be used to identify the proteins in a sample).
How does myoglobin differ to haemoglobin and how is it similar?
Similar = both have a His which brings an Fe2+ ion into the ring, which affects the molecules affinity for oxygen (when O2 is bound, His brings Fe2+ into the plane)
Differ = Myoglobin binds to oxygen tightly, and releases very little to tissues
Haemoglobin releases a lot more oxygen to the tissues, in a cooperative manner
What is the structure of Haemoglobin and how does it’s structure change to allow oxygen binding?
Tetramer - 2 alpha and 2 beta subunits
Has 2 conformations:
- T (tense conformation) when no/little
oxygen is bound (deoxyhaemoglobin)
- R (relaxed conformation) when oxygen
is bound (oxyhaemoglobin)
The T state has a gap in between the 4 subunits, which closes when haemoglobin changes conformation to the R state when oxygen binds
What is the concerted model of haemoglobin cooperative binding, and how does this affect which conformation is favoured?
The 2 states (T+R) of haemoglobin are in equilibrium and that O2 binding stabilises one conformation over the other
The R state will be favoured when there’s more O2 bound (3 or more subunits O2 bound), and the T state will be favoured when there’s less O2 bound (1 or less subunits O2 bound)
The T state will favour having less O2 bound (less subunits with O2 bound), and the R state will favour having more O2 bound (more subunits with O2 bound)
What is the Bohr affect on haemoglobin?
Even a small change in pH is enough to decrease the affinity of haemoglobin to oxygen slightly
What is sickle cell anaemia caused by and how does this affect haemoglobin molecules?
Caused by a mutation where a glutamic acid residue is swapped with the hydrophobic residue Val 6. Val6 is found on the surface of the T (deoxygenated) state, but not the R (oxygenated) state of haemoglobin.
So, mutated deoxyhaemoglobin molecules with the Val6 mutation “sticks” to other hydrophobic residues Phe85 and Val88 on another haemoglobin molecule.
This causes long polymers of haemoglobin molecules to form, and causes red blood cell shapes to change from a round shape to a long chain (sickle shape)
This prevents oxygen being delivered as easily to tissues
What is a treatment option for sickle cell anaemia?
Can drive haemoglobin to be more like myoglobin, which would make haemoglobin always be in it’s R state (bound tightly to oxygen). This would mean that the haemoglobin molecules couldn’t stick together, as there wouldn’t be as many of them in the deoxygenated state to bind together
What are proteases, what are serine proteases and how are they specific?
Protease = enzymes which catalyse peptide bond hydrolysis (breaking up protein chains)
Serine protease = protease which uses an OH as the attacking group
Specific - different serine proteases are specific to different types of peptides
What are 3 examples of serine proteases, and what type of peptides are they specific to and why?
- Chymotrypsin = Has an empty binding pocket (active site), allows aromatic/long non-polar side chains to bind
- Trypsin = contains negative amino acid groups in the binding pocket and so is specific to positively charged amino acid residues binding
- Elastase = contains Valine chains to block up the pocket, so only small molecules can bind
