Transcript File - Elko Science

Intended Learning Objectives
You should be able to…
1. Give 3 examples of proteins that are important to
humans and are currently produced by transgenic
organisms.
2. Describe a protein’s composition.
3. Recognize the 4 basic protein structures.
4. Explain the following key terms: Hydrophobic and
Hydrophilic.
5. Honors: Identify the parts of an amino acid.
Resources: Chapter 3 (3.14) and Notes
Intended Learning Objectives
You should be able to…
6. Explain how we will be able to
isolate the GFP protein for
purification.
Resources: Lab Handouts and Notes
Intended Learning Objectives
You should be able to…
6a. Explain what each of the following
do in order to help extract the GFP.
- Lysozyme
- Freezer
- Centrifuge (1st and 2nd uses)
Intended Learning Objectives
You should be able to…
6b. Explain what each buffer did to the
proteins in the column. Be sure to indicate
the concentration of salt in each.
- Equilibration buffer
- Binding buffer
- Wash buffer
- Elution buffer
Hydrophobic Interactions
Supernatant
Low salt buffer
Protein Structure
o
1
Primary
o
2 Secondary
o
3 Tertiary
o
4 Quaternary
1° Primary Structure
• Linear sequence of amino acids that
make up the polypeptide chain.
• Determined by the genetic code in
particular the mRNA
• The bond between two amino acids is
a peptide bond. This bond is formed by
the removal of a H2O molecule from two
different amino acids, forming a
dipeptide.
• The sequence of amino acids
determines the positioning of the
different chemical groups of each
building block which determines which
groups will interact with each other and
therefore determines the final structure
and function of the molecule.
2° The secondary structure
• coiling of the peptide chain
into a helix or some other
regular pattern of twists or
kinks of the polypeptide chain.
• due to hydrogen bonds
forming between the atoms of
the amino acid backbone of the
polypeptide chain.
• The two most common types
of secondary structure are
called the alpha helix and beta
pleated sheet.
3° Tertiary structure
• three dimensional globular structure formed by
bending and twisting of the polypeptide chain.
•folding of the polypeptide chain is stabilized by
multiple weak, non-covalent interactions. These
interactions include:
+ Hydrogen bonds - form when a Hydrogen atom is
shared by two other atoms.
+ Electrostatic interactions - occur between charged
amino acid side chains.
+ Hydrophobic interactions - amino acids with a polar
(water soluble) side chain are often found on the surface of the
molecule while amino acids with nonpolar (water insoluble) side
chain are buried in the interior. This means that the folded protein
is soluble in water or aqueous solutions.
+ Disulfide Bonds: The polypeptide chains of some
proteins are linked by disulfide bonds.
4° Quaternary structure.
• Proteins that contain more
than one polypeptide chain
• Each polypeptide chain in
the protein is called a subunit.
• The subunits can be the
same polypeptide chain or
different ones.
• For some proteins,
quaternary structure is
required for full activity
(function) of the protein.
Hydrophobic interaction chromatography
HIC
The background
– hydrophobic compounds stick together
– association affected by:
salt concentration  greater concentration =
more hydrophobic interactions
Hydrophobic region
The idea
– stick GFP to a polymer sphere with hydrophobic groups
– wash it off by changing the salt concentration ( decrease or
increase?)
True or False
1. The function of a protein is determined by its
structure/shape. TRUE
2. Hydrophobic molecules tend to “hang out”
or bind to other hydrophobic molecules. TRUE
3. Higher concentrations of salt decrease the
binding of proteins to hydrophobic polymers.FALSE
4. Molecules that are Hydrophobic are polar
molecules.
FALSE
5. Electrostatic attractions between amino acids
occur in the primary structure of a protein. FALSE
Which of the following is the
strongest bond?
• Polar covalent
• Nonpolar covalent
• Hydrogen
• Electrostatic
Which protein structure does the picture
depict below?
a-helix or beta sheet?
a-helix
Primary, secondary, tertiary, or
quaternary structure?
secondary
Which of the following
weakened the cell wall of
the bacteria cells.
• lysozyme
• freezer
• centrifuge
• arabinose
Which of the following had the
highest salt concentration?
• Wash buffer
• Equilibration buffer
• Elution Buffer
• Binding Buffer
Hydrophobic Interaction Chromatography
Biopolymer (phenyl agarose - Binding Surface)
Driving force for hydrophobic adsorption
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.
Temperature plays a strong role
Hydrophobic region
Ammonium sulfate, by virtue of its good salting-out
properties and high solubility in water is used as an
eluting buffer
(Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)
Hydrophobic interaction chromatography (HIC)
separates molecules based on their
hydrophobicity. Molecules that contain both
hydrophobic and hydrophilic regions are
applied to an HIC column in a high-salt buffer.
The salt in the buffer reduces the solvation of
the sample. As overall solvation decreases,
hydrophobic regions that become exposed are
adsorbed by the medium. The more
hydrophobic the molecule, the less salt is
needed to promote binding. Usually a
decreasing salt gradient is used to elute
samples from the column in order of increasing
hydrophobicity.