Introduction into Cell Biology 3 The building blocks of life - Proteins - PowerPoint PPT Presentation

Loading...

PPT – Introduction into Cell Biology 3 The building blocks of life - Proteins PowerPoint presentation | free to download - id: 448823-NDIzN



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Introduction into Cell Biology 3 The building blocks of life - Proteins

Description:

Introduction into Cell Biology 3 The building blocks of life - Proteins * * * * * We will talk about 2 subjects today: X-ray crystallography Protein purification * 3D ... – PowerPoint PPT presentation

Number of Views:210
Avg rating:3.0/5.0
Slides: 32
Provided by: EP91
Learn more at: http://homes.nano.aau.dk
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Introduction into Cell Biology 3 The building blocks of life - Proteins


1
Introduction into Cell Biology 3The building
blocks of life - Proteins
2
  • We will talk about 2 subjects today
  • X-ray crystallography
  • Protein purification

3
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 -gt out of the 35000
protein structures solved, X-ray crystallography
is responsible for 29000. NMR spectroscopy has
contributed almost 5000.
4
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

5
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.
6
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

7
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 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.
A common method for crystallisation is hanging
drop vapour diffusion.
8
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 lt 4
angstroms 400 picometers).
9
Structure determination by X-ray crystallography
X-ray beam hits crystal -gt constructive
interference between diffracted X-rays that are
in-phase reinforce each other -gt so that the
diffraction pattern becomes detectable.
10
Diffraction pattern of the crystal
11
Electron Density Map of Crystal -gt Atomic
Structure
12
3D Structure of Proteins Different
Visualizations
13
Protein Purification
What is protein purification? Isolation of one
specific type of protein from a pool of many
different proteins (produced in a cell)
14
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

15
Processes that can be Used to Purify Proteins
Separation
Process Basis of
Separation Precipitation
ammonium sulfate
solubility isoelectric solubility,
pI Chromatography gel filtration
(SEC) size, shape ion
exchange (IEX) charge, charge
distribution hydrophobic
interaction(HIC) hydrophobicity affinity
binding
site immunoaffinity (IAC) specific
epitope chromatofocusing
pI Electrophoresis gel
electrophoresis (PAGE) charge, size,
shape isoelectric focusing (IEF)
pI Centrifugation sucrose
gradient size shape, density Ultrafiltration
ultrafiltration (UF)
size, shape
16
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.
17
In the animation below the red molecules are more
soluble in the liquid (or less volatile) than are
the green molecules.
18
Kinds of Chromatography
1. Liquid Column Chromatography   2. Gas Liquid
Chromatography   3. Thin-layer Chromatography
19
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.
20
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)
21
Major Chromatographic Methods in Protein
Purification
22
Protein purification can be a multi-step procedure
23
Usually requires use of complex equipment
24
Column Chromatography
  • Most common method for separating proteins

25
Size-exclusion chromatography
Separation properties -gt size
26
Size-exclusion chromatography
Absorbance at 280 is used to identify
protein-containing fractions. You can also
perform an enzyme specific assay.
27
Ion-Exchange chromatography
Separation properties -gt Charge
If pH mobile phase 7.2 Then charge of the
proteins (-) (-)
() ()
Anion exchange column charged
28
Ion-Exchange chromatography
-
-


Increased salt concentration
29
Hydrophobic Interaction Chromatography
Separation Properties -gt 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
30
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

31
Questions?
About PowerShow.com