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Structural Biology > Structural Biology - Protein Seperation > Flashcards

Structural Biology - Protein Seperation Flashcards

1
Q

What is chromatography used for?

A

Protein purification/seperation

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2
Q

What is electrophoresis used for in protein separation?

A

Monitor how separation is going visually (NOT a purification method)

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3
Q

What’s the history of protein purification?

A

proteins initially purified from natural sources only, then from recombinant host cells

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4
Q

What are proteins purified in and why?

A

A recombinant host (E.coli most commonly used)
designed to overexposes the protein, in a folded functional form

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5
Q

What gives proteins unique surface properties?

A

Unique shape, structure and amino acid sequence. Proteins fold into a unique shape and display heterogenous surface charge and hydrophobicity

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6
Q

What properties of proteins can be exploited for purification?

A

Positively charged residues, negatively charged residues, hydrophobicity, ligand binding sites

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7
Q

Why do we purify proteins?

A

Enzyme kinetics
Enzyme regulation and inhibition
Macromolecular complexes they form
Structural analysis (EM, X-Ray, crystallography, NMR)
Drug discovery and biologics, diagnostics, antibody production

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8
Q

Explain process of salting out?

A

Uses differing surface hydrophobic properties of proteins for crude separation

Two different environments of water around a protein: Ordered and bulk
The ordering of water molecules around hydrophobic patches (hydrophobic effect) is unfavourable entropically
Hydrophilic aa interact with bulk water to form H-bonds

Salt is added and solvated in the bulk region: water interacts with salt instead of protein

Proteins form hydrophobic interactions with each other

Aggregation and precipitation of protein

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9
Q

What is the Hoffmeister series?

A

Shows ranking of ions toward their ability to precipitate a mixture of proteins
ammonium sulphate is commonly used as a salt for salting out

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10
Q

What is adsorption/desorption?

A

Solid/liquid phase
Proteins in solution binding differentially to the column (solid phase) depending on the thermodynamics

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11
Q

What is permeation?

A

Liquid/liquid phase
dependent on the rate of diffusion (kinetics) between liquid phases. No interaction with the solid phase

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12
Q

What is the experimental setup of protein chromatography?

A

glass tube
solid gel/stationary phase (selective as used for separation)
highly specific interactions
buffer solution
fraction collector

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13
Q

What things can differ in the setup of protein chromatography?

A

selectivity of stationary phase (separate based on charge/size)
hydrodynamics (liquid flow and solid phase)
column design (size and packaging)

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14
Q

What factors does the Langmuir isotherm consider?

A

Fraction of surface sites occupied (0-1)
concentration of binding sites
dissociation constant (the lower, the tighter the binding

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15
Q

Langmuir isotherm: what is good binding (adsorption) dependent on?

A

High concentration of binding sites
low dissociation constant
alpha must be 0.8-1 for adsorption

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16
Q

What factors affect Kd and alpha in the Langmuir isotherm?

A

pH (protein charge)
I (ionic strength)
Polarity

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17
Q

What value of alpha gives desorption the Langmuir isotherm?

A

once protein is bound to column it must be mobilised again
alpha must be 0.5 or less

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18
Q

What affects alpha in the Langmuir isotherm?

A

alpha depends on Kd
Vary Kd from 0.01mM to 1mM for elution to change alpha from 1 to 0

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19
Q

What are assumptions regarding the Langmuir isotherm?

A

adsorption sites are not all of equal strength so there is a range of Kds
Protein concentration is not constant within a protein band (diffusion)
Ionic strength, hydrophobicity and multipoint binding are ignored
Concentration of binding sites varies

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20
Q

Is adsorption/desorption an instantaneous process?

A

No
protein needs some time in contact with the solid phase to reach equilibrium
too fast = no time to interact
too slow = loose resolution and diffusion of protein

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21
Q

Explain the dynamics of proteins binding to a column

A

Proteins are always binding + coming off the column
Dilution occurs as proteins flow down the column
Dispertion (spreading of protein band)

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22
Q

What are the two contributors to dispersion in a column?

A

Turbulent flow (too fast)
Diffusion (too slow)

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23
Q

Explain the process of diffusion

A

When flow is too slow
column is filled with buffer and contains stationary phase
protein mixture is added
buffer is added over the top so protein moves down the column
regions above and below the protein band contain to protein so molecules diffuse into these regions (band spreading)

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24
Q

What is typically used to measure protein concentration

A

Fluorescence or UV (aromatic residues in proteins)

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25
Q

Explain band spreading (diffusion) as a Gaussian shaped profile

A

highest protein concentration in the middle
loss of protein concentration at the edges
occurs when column moves too slowly
protein concentration vs volume graph
Dispertion = volume width of peak at half height
resolution = separation of two peaks over the average of dispersed widths

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26
Q

Explain the process of turbulent flow

A

When flow is too fast
Solution passes through channels between particles of solid phase. These are uneven so turbulence occurs when flow is too fast

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27
Q

What happens when the flow is too fast in a column?

A

Reduces diffusional broadening
Causes turbulent flow and equilibrium may not establish

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28
Q

Explain the process of self sharpening in chromatography

A

alpha (the fraction of protein bound to the column) changes as protein diffuses.
Lower protein concentration at top and bottom edges of the band = increased likelihood of binding (alpha increases) and slower movement down the column
Centre of the band is moving fast (high concentration) and edges are moving slow = sharpening of leading edge of the band and tailing of the rear edge of the band
lower resolution in the trailing edge

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29
Q

How does alpha change with protein concentration?

A

alpha (fraction of protein bound) increases as protein concentration decreases

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30
Q

List the different types of adsorption/desorption

A

Ion exchange chromatography
Chromatofocussing
Hydrophobic interaction chromatography
Affinity chromatography

31
Q

What properties does the solid phase have in chromatography?

A

Hydrophilic to minimise non-specific binding (usually carbohydrate based)
highly porous to avoid size exclusion
can be derivatised
rigid to minimise compression under flow
stable to extremes of pH
capable of being made into fine particles

32
Q

What are the typical substituents used in IEC?

A

A Positively charged group: ex. ammonium. Weak base is protein is highly charged and strong base is protein is slightly charged.
A negatively charged group (acid)

33
Q

How does ion exchange work?

A

Proteins are loaded onto column with low ionic strength.
pI is the point where the proteins no charge. Above the pI, protein has -ve charge so binds to a +ve anion exchanger. Below the pI protein has +ve charge so binds to -ve cation exchanger.
To release/elute protein, change pH to pI OR increase ionic strength/salt concentration (small ions like NaCl compete for the charge on the protein and column and increase the kD)

34
Q

What happens to alpha when the charge on the protein does not change in IEC?

A

Alpha titrates with the ratio of charged (binding) to uncharged (non-binding) groups.
Titration of protein groups is more important than the binding sites and this will affect kD

35
Q

How many charges are required to allow for elution?

A

Each favourable charged (ionic) interaction is 1-2kJ mol^-1
ΔG = -RT ln K
So each charge of 6kJ mol^-1 alter K by a factor of 10
The same as a change in protein charge of 4 switches between binding and desorption

Assume Г = 0.5 mM and Kd = 0.05 mM gives α = 0.91
Changing Kd from 0.05mM to 0.5mM (factor of 10) gives α = 0.5 which allows elution

36
Q

What happens when you overload an IEC column?

A

Fractionation my size and not charge
column is porous, so overfilling would close the pores and prevent molecules from moving down the column

37
Q

How does the pH vary in the IEC column and how can this be solved?

A

pH at the surface may be different from bulk solution, as H+ is excluded and +ve and concentrated at -ve.
pH can affect protein stability
Use a buffer to minimise pH gradient.
But need to make sure that buffer does not bind preferentially hence changing the pH

38
Q

What does the modern Ion exchange system look like?

A

2 buffers: A (low salt) and B (high salt)
flow through valve which introduces the protein and goes into column
introduce buffer B high salt over a linear gradient
Fraction collector is a series of tubes that collects amounts from column
UV detector to measure where the protein is in the fraction collector

39
Q

How does band sharpening work in IEC with salt?

A

A linear gradient of salt is introduced into the column.
Rear edge has high salt, front has low salt.
High salt = more elution/less binding so increased kD and decreased alpha.
so trailing/rear edge catches up with leading edge = improved resolution

40
Q

What is chromatofocussing and what is it limited by?

A

Variation of band sharpening but uses pH gradient instead of salt gradient.
In a well optimised column resolution is limited by diffusional broadening.
try to get the protein to rebind to allow the trailing edge to catch up

41
Q

Explain the process chromatofocussing in a column?

A

Use an anion exchange resin with protein pI 7.
pH gradient down the column is very steep (not like salt linear gradient)
Column starts at pH 9, so protein binds
Elution occurs when pH is close to pI at bottom of column (protein is most concentrated here)

42
Q

How do we create a pH gradient in a column for chromatofocussing?

A

adding acid does not work
use exchange and buffer with range of pKa values and constant buffering capacity
ex. polyaminopolycarboxylic acids (ampholytes)
So proteins elute in order of their pIs

43
Q

What is a hydrophobic interaction column (HIC)?

A

Uses hydrophobic interactions to separate proteins (as proteins with similar pI are difficult to separate)
Solid matrix carried hydrophobic groups (phenyl/octyl).
Start with high salt and gradient down (opposite to IEC) = salting out effect
Highly hydrophobic protein can be eluted with organic solvent/H2O mixture (ethylene glycol)
This method is a last resort (IEC should be used first) because it can impact protein stability

44
Q

What is reverse phase chromatography?

A

Similar to hydrophobic interactions.
Solid matrix carries a more hydrophobic ligand like C18 or biphenyl.
Under high pressure
Gives high resolution
Proteins are denatured so not used for soluble proteins (only for peptides/carbohydrates)

45
Q

What is affinity chromatography?

A

Separation based on specific binding between immobilised ligand and protein

46
Q

What are some common issues with affinity chromatography?

A

non specific binding at high protein concentrations
high cost of affinity matrices in industry
lower capacity of immobilised ligand (adding too much protein is bad)

47
Q

When is affinity chromatography used?

A

Usually used in combination with other methods for ‘polishing’ steps

48
Q

What types of ligands can be used for affinity chromatography?

A

substrate analogues/inhibitors for enzymes.
cofactors ex. NAD+
lectins - bind glycoproteins
Dye ex. blue dye dextran
antibodies
fusion proteins ex. GST, poly-His, MBP fusion at N or C terminus

49
Q

What is the role of CNBr in affinity chromatography?

A

CNBr is a pre-activated resin
CNBr reacts with OH groups on matrix to form cyanite esters.
These groups react with amine groups of ligands to form a covalent bond which immobilises the ligand.

50
Q

How does elution work in affinity chromatography?

A

Protein binds to immobilised ligand in the column. Elute by using soluble ligand. Protein becomes more mobile and moves down the column. Works well for lectins and fusion proteins.
Can increase concentration of ligand in linear gradient OR in one step.

Can also change I (ionic strength) or pH as binding is dependent on these

51
Q

What is gel filtration?

A

Polishing method used last due to low resolution and limited capacity.
Liquid-liquid phase
Column has a range of pore sizes
Largest molecules come out first (pass through the side instead of the pores)
Water and small molecules come out last (passes through all the pores)
No interaction with solid phase (it is inert)
No thermodynamic effect (kD) so resolution is low
Limited capacity and mixture needs to be concentrated (<10mg/ml)
Long thin columns the best

52
Q

What does a chromatograph from gel filtration look like?

A

See notes for image
V0 = volume outside all the pores, any protein can go through this
Vt = total volume
Ve = elution volume, where separation occurs

53
Q

What is a calibration curve used for in gel filtration?

A

Plots Kav vs log Mr in a straight line
Used to buy appropriate column for the Mr

54
Q

Explain multi step purification process and the purification table

A

Every step leads to a loss in yield
Purification table used to see how efficient each step is
Activity is only for enzymes
loss of total units but increase in specific activity between each step
purification factor used to determine efficiency of each step

55
Q

What is protein electrophoresis?

A

Migration of proteins in an electric field
Polymeric medium
Diffusional broadening still present but reduced as molecules diffuse slower in gel

56
Q

What does the rate of migration depend on Electrophoresis?

A

Field strength (voltage)
ionic strength (buffer with higher ionic strength =conducts current more = slower migration
net charge
temperature
molecular size and shape
viscocity

57
Q

what are the 4 types of electrophoresis?

A

Denaturing condition - migration depends on size
Native conditions - migration depends on size and charge
Isoelectric focussing - migration dependent on pI
2D - migration depends on pI in one dimension and size in the second

58
Q

Explain the polymer phase (acrylamide) in gels

A

Acrylamide cross linked with methylene bis-acrylamide
Ratio of acrylamide to methylene bis-acrylamide and concentration determines pore size (which determines rate of separation)
5% acrylamide = no Mw sieving
10-15% = proteins are separated by size

59
Q

How are proteins detected in gel elecrophoresis?

A

Coomassie blue dye or silver stain

60
Q

How do SDS-PAGE gels work?

A

proteins denatured by heating to 100 degrees
reduction agent (SDS/meta mercapoethanol) used to reduce disulphide bonds
SDS binds hydrophobic parts on protein and unfolds polypeptide
SDS is anionic (negatively charged) so now each protein has the same negative charge density
proteins migrate according to size

61
Q

What is a discontinuous gel?

A

Acrylamide between two plastic/glass plates
Anode at bottom, cathode at top
Tris-glycine buffer at the top and bottom
Top gel (stacking gel) 5% acrylamide pH is 6.7 (All proteins will have the same mobility due to large pores)
Second gel (running gel) acrylamide 10-15% acrylamide pH is 8.9 (Molecular sized pores that separate proteins based on size)
Smaller proteins run faster than larger proteins

62
Q

Explain how glycine is used in the stacking and running gels

A

pH 6.7: slightly net negative charge (pI is 6) in stacking gel
pH 8.9: large negative charge (above pI) in running gel

Rate of migration = V x (δMx)
δ is the proportion of charge molecules
Mobility is slow when glycine is close to its pI at pH 6.7
Whole population moves as a group (share the small delta negative)

63
Q

How does a boundary form between glycine and chloride ions in the stacking gel (pH 6.7)?

A

Glycine moves slower than chloride
Forms a conductivity gradient (glycine low conductivity+small neg. charge, chloride high conductivity+full neg.charge)
Constant current is applied so voltage increases in glycine region
Glycine now moves faster
Proteins are trapped between two regions
Creates a concentrated/narrow protein band
reduces human error when pipetting
After in running gel: pH increases and glycine is full charged so proteins are sieved by Mr

64
Q

How can electrophoresis be used to monitor purity before/after chromatography?

A
  1. homogenate with proteins
  2. IEC more pure sample
  3. Affinity chromatography pure protein (should show one band)
  4. Marker
65
Q

How does native PAGE work?

A

Similar to SDS-PAHGE but no denaturing
Uses intrinsic charge or protein: most proteins have pI 4-7 and native gels run at pH 8-9.5 so most proteins will have net negative charge
Charged dyes can be used
The closer the pH is to the pI, the longer the running time

66
Q

How can an oligomeric protein be identified with gel electrophoresis?

A

Run denaturing (only size) and non denaturing (separation based on size and charge) at the same time. If the band is shifted up, it is oligomeric. Denaturing will show multiple bands, non-denaturing keeps native interactions so will show a larger band.

67
Q

How does isoelectric focusing work?

A

Uses intrinsic charge (no denaturing)
Migration through a preformed pH gradient across the gel
Proteins migrate to their pIs (net charge=0) and focus there (stop migrating)
very high resolution <0.1pH units

68
Q

How is a pH gradient achieved in isoelectric focusing?

A

Ampholytic buffers
Are polyomino polycarboxylic acids
Have a range of pKa’s
Have ionisable negative and positive groups
Move as a group toward the cathode or anode
Separate them out to create a buffer system

69
Q

How does 2-D electrophoresis work and what does it allow?

A

Separate hundreds of proteins
Migration through a pH gradient first, then rotate field and migrate through SDS gel
Get pI vs Molecular weight
High resolution

70
Q

What are the advantages of capillary electrophoresis?

A

easy and predictable selectivity
high separation efficiency
small sample sizes
fast seperations
can be automated and coupled to mass spec
quantitation

71
Q

What are the disadvantages of capillary electrophoresis?

A

low concentrations and large volumes difficult for sticky molecules
reproducibility problems

72
Q

How does capillary electrophoresis work?

A

Occurs in negatively charged capillary
High surface area to volume ratio
Electric field is applied along the capillary
Many positive charges near negative wall of the capillary
Net flow is towards the cathode; even though anions are moving the other way the force of the positive charges is stronger
Occurs via electroosmotic flow
Molecules migrate according to Mw
Data is displayed as an electropherogram

73
Q

How does capillary Isoelectric focusing work?

A

IEF separates proteins based on their pI
Electroosmotic flow is suppressed by coating it with teflon
Capillary is filled with a buffer to create a pH gradient
Electric field is applied
Molecules move to pH where their net charge is 0 (pI) and focus there
After focusing, zone is mobilised by pressure or chemical means (add salt to compete with H+/OH-)

Structural Biology (6 decks)

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