Introduction into Cell Biology 3

The building blocks of life - Proteins

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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.

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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 (N 15 , C 13 ) - 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 compound of interest. This is because a single molecule in solution has insufficient scattering power alone. crystallize the 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 common method for crystallisation is

hanging drop vapour diffusion

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A small droplet of concentrated protein- and precipitate-containing solution is applied to a glass coverslip which is then inverted so 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 picometers ).

angstroms = 400 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 .

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Diffraction pattern of the crystal 10

Electron Density Map of Crystal -> Atomic Structure

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3D Structure of Proteins – Different Visualizations

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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?

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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 Process Precipitation

ammonium sulfate isoelectric

Basis of Separation

solubility solubility, pI

Chromatography Electrophoresis Centrifugation Ultrafiltration

gel filtration (SEC) size, shape ion exchange (IEX) charge, charge distribution hydrophobic interaction(HIC) hydrophobicity affinity immunoaffinity (IAC) chromatofocusing binding site specific epitope pI gel electrophoresis (PAGE) charge, size, shape isoelectric focusing (IEF) pI sucrose gradient ultrafiltration (UF) size shape, density size, shape 15

Ch rom ato gra phy 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.

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In the animation below the red liquid molecules are more soluble in the (or less volatile) than are the green molecules. 17

Kinds of Chromatography

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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 OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

Solvent(mobile or moving phase)

A + B + C

Sample (A+B+C) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Column OOOOOOOOOO OOOOOOOOOOO Solid Particles OOOOOOOOOOO (packing material- OOOOOOOOOO OOOOOOOOOOO stationary phase) OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Eluant (eluate) 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 Gel Filtration Hydrophobic Interaction 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 protein containing fractions. You can also perform an enzyme specific assay.

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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 + + + + + + + Na+ Na+ Na+ + Increased salt concentration + + + Na+ Na+ Na+ Na+ Na+ Na+ + + + Cl Cl + Cl + + Cl + Cl 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:

 Ni 2+  binds to poly Histines (example 6xHis)  Specific antibodies (anti-Flag tag)  glutathione  binds to GST  Protein A or G  binds antibodies 30

Questions?

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