Chromatography

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Introduction into
Cell Biology 3
The building blocks of life - Proteins
1
We will talk about 2 subjects today:
- X-ray crystallography
- Protein purification
2
3D Structure Determination
There are two methods used:
- X-ray crystallography
- Nuclear Magnetic Resonance (NMR)
Biological X-ray crystallography is to date the most prolific discipline
within the area of Structural biology;
-> out of the ~35000 protein structures solved, X-ray crystallography
is responsible for ~29000. NMR spectroscopy has contributed almost
5000.
3
Structure determination by NMR
Advantage:
-Structure of Protein
in solution
- Flexible regions in
protein can be
detected
Disadvantage:
-Size limitation (30-50
kDa)
- stable isotope
labeling (N15, C13)
- high protein
concentration in
solution
4
Structure determination by
X-ray crystallography
The first protein crystal structure was of sperm whale myoglobin, as
determined by Max Perutz and Sir John Cowdery Kendrew in 1958, which led to
a Nobel Prize in Chemistry.
In order to solve a crystal structure, you must first crystallize the
compound of interest. This is because a single molecule in solution has
insufficient scattering power alone.
A crystal can be considered to be an (effectively) infinite repeating
array of our molecule of interest.
5
Structure determination by
X-ray crystallography
Crystallisation of macromolecules is not trivial. Traditional methods of crystallising
inorganic molecules have been modified to be gentle enough for proteins, which are
sensitive to temperature and high concentrations of organic solvents.
Many methods exist to crystallise proteins, but the two most successful methods
are the microbatch and vapour diffusion techniques.
Concentrated solutions of the protein are mixed with various solutions, which
typically consist of:
- a buffer to control the pH of the experiment
- a Precipitating agent, to induce supersaturation (typically Poly ethylene glycols, Salts such as
Ammonium sulphate or organic alcohols).
- other salts or additives, such as detergents or co-factors
6
Structure determination by
X-ray crystallography
A small droplet of concentrated
protein- and precipitate-containing
solution is applied to a glass
coverslip which is then inverted so
A common method for crystallisation is
hanging drop vapour diffusion.
as to suspended the droplet above
a larger reservoir of a similar
solution lacking protein but
containing a higher concentration of
precipitate, and the chamber
sealed.
Over time, the droplet containing
protein equilibrates with the larger
reservoir beneath it as volatile
water in the droplet leaves the
droplet and transfers to the
reservoir, effectively increasing
the precipitate concentration in the
protein droplet. In solutions of a
favourable composition, the protein
becomes supersaturated and
crystal nuclei form, leading to
crystal growth.
7
Structure determination by
X-ray crystallography
That is the optimal outcome. Otherwise (and typically) the protein
forms a useless and amorphous mass as protein precipitates out of
solution. Typically protein crystallographers can screen hundreds or
thousands of conditions before a suitable condition is found that
leads to a crystal of suitable quality (Resolution < 4 angstroms = 400
picometers).
8
Structure determination by X-ray
crystallography
X-ray beam hits crystal
-> constructive interference
between diffracted X-rays
that are in-phase reinforce
each other
-> so that the diffraction
pattern becomes detectable.
9
Diffraction pattern of the crystal
10
Electron Density Map of Crystal -> Atomic
Structure
11
3D Structure of Proteins –
Different Visualizations
12
Protein Purification
What is protein purification?
Isolation of one specific type of protein from a
pool of many different proteins (produced in a
cell)
13
Why purify proteins?
 Purified





proteins can be used to:
Study enzymatic functions and enzymatic
regulation
Study protein interactions
Produce antibodies
Protein for Biosensors
Perform structural analysis by x-ray
crystallography and NMR spectroscopy
14
Processes that can be Used to Purify
Proteins
Separation
Precipitation
Process
ammonium sulfate
isoelectric
Basis of Separation
solubility
solubility, pI
Chromatography
gel filtration (SEC)
ion exchange (IEX)
hydrophobic interaction(HIC)
affinity
immunoaffinity (IAC)
chromatofocusing
size, shape
charge, charge distribution
hydrophobicity
binding site
specific epitope
pI
Electrophoresis
gel electrophoresis (PAGE)
isoelectric focusing (IEF)
Centrifugation
sucrose gradient
size shape, density
Ultrafiltration
ultrafiltration (UF)
size, shape
charge, size, shape
pI
15
Chromatography
Is a technique used to separate and identify the
components of a mixture.
Works by allowing the molecules present in the mixture to
distribute themselves between a stationary and a mobile
medium.
Molecules that spend most of their time in the mobile phase are
carried along faster.
16
In the animation below the red molecules are more soluble in the
liquid (or less volatile) than are the green molecules.
17
Kinds of Chromatography
1. Liquid Column Chromatography
2. Gas Liquid Chromatography
3. Thin-layer Chromatography
18
Liquid Column Chromatography
A sample mixture is passed through
a column packed with solid
particles.
With the proper solvents, packing
conditions, some components in the
sample will travel the column more
slowly than others resulting in the
desired separation.
DIAGRAM O F S IMPLE LIQ UID C O LUMN C HRO MATO G RAPHY
Solvent (m obile or
moving phase)
A+ B+C
Sam ple
(A+B+C)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
Column
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
Solid P articles
OOOOOO OOOOO (packing materialOOOOOO OOOO stationary phase)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOA OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOB OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOC OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
Eluant (eluat e)
19
FOUR BASIC LIQUID CHROMATOGRAPHY
The 4 basic liquid chromatography modes used for protein
purification:
1. Adsorption chromatography (Affinity chromatography,
hydrophobic interaction)
2. Partition chromatography (Reverse phase)
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
20
Major Chromatographic Methods in Protein Purification
Hydrophobic Interaction
Gel Filtration
Ion Exchange
Affinity
Reverse Phase
21
Protein purification can be a multi-step
procedure
22
Usually
requires use of complex
equipment
23
Column Chromatography
Most common method for separating proteins
24
Size-exclusion chromatography
Separation properties -> size
25
Size-exclusion chromatography
Absorbance at 280 is used to identify proteincontaining fractions. You can also perform an
enzyme specific assay.
26
Ion-Exchange chromatography
Separation properties -> Charge
-
-
+
+
(-)
(-)
(+)
(+)
If pH mobile phase =7.2
Then charge of the proteins:
Anion exchange column = + charged
++
++
+
++
+
-
-
-
-
+
+
++
+
++
+
+
+
27
Ion-Exchange chromatography
-
+
-
+
+
-
-
+
+
+
+
+
Cl- +
+
Cl-
+
Cl-
+
+
+ Cl-
+
+
Na+ Na+ -
+ Cl-
Na+
Na+ -
Na+Na+
+
Increased salt concentration
Na+
Na+ Na+
Cl-
+
28
Hydrophobic Interaction Chromatography
Separation Properties -> Hydrophobicity
Driving force for hydrophobic adsorption are
Water molecules surround the analyte and the binding
surface.
When a hydrophobic region of a biopolymer binds to
the surface of a mildly hydrophobic stationary phase,
hydrophilic water molecules are effectively released
from the surrounding hydrophobic areas causing a
thermodynamically favorable change in entropy.
Ammonium sulfate, by virtue of its good salting-out
properties and high solubility in water is used as an
eluting buffer
Hydrophobic region
29
Affinity chromatography

Commonly used affinity
columns:

Ni2+  binds to poly Histines
(example 6xHis)

Specific antibodies (anti-Flag tag)

glutathione  binds to GST

Protein A or G  binds antibodies
30
Questions?
31
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