Download protein structure and function

PROTEIN STRUCTURE AND
FUNCTION
Proteins
Are Where It’s At
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Proteomics
Gene regulation
Drug Discovery
Understanding evolution
Etc.
Proteins are
Where It’s Been
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Enzymes
ß-galactosidase
Antibodies
Anti-Hepatitis B
Hormones
Human Growth Hormone
Estrogen
Testosterone
Proteins are
Where It’s Been
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Structural proteins
Collagen
Transportation
Haemoglobin
Proteins Are Us
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In cells, when something needs to be done, it
is a protein that does it.
The human body contains over 30,000
different types of protein
Other organisms have many of the same
proteins as well as different ones
Enzymes are the biggest class
3,000 enzymes in average mammalian cell
ß-galactosidase is an enzyme
Classes of Proteins
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Structural
Collagen
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Found in bone
and skin
Keratin
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Makes hair and
nails
Fibrin
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Helps clot blood
Elastin
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Major part of
ligaments
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Transcription Factors
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Control expression
of genes
Hormones
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Control body
function
Antibodies
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Fight infection
Enzymes
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Speed up
chemical reactions
Carrier molecules
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Haemoglobin carries oxygen in
the blood
Proteins Are
Diverse In Structure
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Proteins can do many things because they are
structurally diverse
Differ in many properties:
Size
Shape
Charge distribution
Hydrophobicity
Solubility properties
Variability Comes
From Amino Acids
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Are polymers composed of 20 different amino
acid building blocks
As letters can be arranged in many ways, so
too can amino acids
Number, type and arrangement of amino acids
determines structure and function
Insulin has about 50 AA
Most are >> bigger - from 100s to 1000s
Allows for great diversity
Amino Acids
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All amino acids have a carboxyl group and an
amino group
A different R group is attached to each amino
acid
Amino Acid
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R groups make each amino acid different
Some are:
Polar
Nonpolar
Charged
Acids
Bases
Twenty Amino acids
Folding
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DNA always has same structure
But proteins fold into many different shapes
Folding depends ultimately on amino acid
composition
Structure of proteins determines function
Structure allows proteins to
BIND to other molecules
RECOGNIZE other molecules
Protein Structure
And Function
Shape Is Critical
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Change in one amino acid can change the
structure of the protein with a large effect on
function
Sickle cell anemia
Sickle Cell Anemia
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Single DNA base pair is mutated
Therefore one amino acid is altered
Glutamic acid is switched to valine
Glutamic acid is negatively charged, valine is
neutral
Changes how hemoglobin packs in cells
Alters shape of red blood cells when oxygen is
low.
Image from
Medline Plus
Levels of Protein
Structure
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Protein structure is complex and important, so
it is classified into:
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1° - Primary
2°- Secondary
3° -Tertiary
4°- Quaternary
Primary Structure
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Linear sequence of amino acids
Peptide bond (covalent bond) holds it
together
Beads on a string
Primary Structure
Peptide Bonds
R O
H R
l
ll
l
l
NH2 –C – C – 0 –H + H –N – C—COOH
l
l
H
H
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R O H R
l ll l
l
NH2 -- C –C – N – C – COOH + H2O
l
l
H
H
Secondary Structure
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Regular repeating patterns of twists or kinks of
the amino acid chain
Examples
Alpha helix
Beta pleated sheet
Secondary Structure
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Hydrogen bonds hold structure together
Weak, noncovalent, molecular interactions
Hydrogen atom is bonded to an
electronegative atom (like F, O, N) that is also
partially bonded to another atom (usually also
F, O, N)
Figure from National Human
Genome Research Institute,
by artist Darryl Leja. Used
with permission.
Tertiary
Structure 3°
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3-D Globular Configuration formed by bending
and twisting of the polypeptide chain
Stabilized by:
Hydrogen bonds
Electrostatic interactions
1. (Positive and negative)
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Hydrophobic interactions
Sometimes covalent bonds
1. Disulfide bonds
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Quaternary
Structure 4°
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Two or more polypeptide chains associate
with each other
ß-Galactosidase
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Link to Protein Data Bank for picture of
molecular image of ß-galactosidase
www.pdb.org
Higher Order
Structure
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Higher order (secondary, tertiary, quaternary)
structure is relatively “weak”
In nature, “weakness” of noncovalent
interactions is important
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Flexibility
1. Enzymes change shape when binding to
their substrates
2. Necessary for proper function
How Proteins Lose
Normal Structure And Function
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Primary structure hard to disrupt; covalent
bonds are strong
How Proteins Lose
Normal Structure And Function
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Can be broken apart by enzymes (proteases)
that digest the covalent peptide bonds
Called proteolysis
Occurs naturally in digestion
Can be a problem in the lab; proteases can
destroy protein of interest
Use cold to avoid proteolysis
How Proteins Lose
Their Structure And Function
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Sulfur groups on cysteines may undergo
oxidation to form disulfide bonds that are not
normally present
Proteins can aggregate leading to precipitation
Proteins can adsorb (stick to) surfaces
Higher Order
Structure In Lab
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Loss of higher order structure is denaturation
Denaturation occurs fairly easily
Affected by changes in pH
Ionic strength
Temperature
May or may not be reversible
Denaturation
Manipulating
Higher Order Structure
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Often manipulated in lab
Destroy folding when we do PAGE
Use buffers to maintain the structure
Use cold temperatures
Add reducing agents to prevent unwanted
disulfide bonds in the lab --DTT or -ME
Analyzing
Protein Structure
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X-Ray Crystallography
Like a CAT scan in medicine
X-ray taken at multiple angles and
computer uses the data to calculate a 3D
image
Nuclear Magnetic Resonance
Like an MRI in medicine
X-ray
Crystallography
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Isolate and purify protein
Form a crystal of the protein
Molecules of the protein are arranged in an
orderly lattice
1. Dissolve protein in solvent
2. Precipitate into a crystal
X-Ray the crystal
X-ray
Crystallography
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Analyze diffraction pattern using software
Make electron density map
Process used to take years
Different versions of crystal for comparison
1. Each with different heavy metal in lattice
to provide reference point
New X-ray sources - synchrotrons have
reduced data collection time to few days
X-ray
Crystallography
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Synchotron - Argonne National Lab
Still takes weeks to go from gene sequence to
3-D structure
(mrsec website, nanotechnology at UWMadison)
Structural
Genomics
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Goal to solve thousands of structures a year
Large scale automation required
Syrrx - structural genomics company
Robot places a drop of protein into 480
wells
11,000 crystallization experiments in 24
hours
New robot - 130,000 a day
Nuclear Magnetic
Resonance
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Similar to MRI - Magnetic Resonance imaging
in medicine
No need for crystals, proteins in solution
Works for relatively simple proteins