PROTEIN STRUCTURE AND FUNCTION

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PROTEIN STRUCTURE AND FUNCTION
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Proteins Are Where Its At

• Proteomics
• Gene regulation
• Drug Discovery
• Understanding evolution
• Etc.

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Proteins are Where Its Been

• Enzymes
• ß-galactosidase
• Antibodies
• Anti-Hepatitis B
• Hormones
• Human Growth Hormone
• Estrogen
• Testosterone

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Proteins are Where Its Been

• Structural proteins
• Collagen
• Transportation
• Hemoglobin

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Proteins Are Us

• In cells, when something needs to be done, it is
  a protein that does it.
• 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 biggest class
• 3,000 enzymes in average mammalian cell
• ß-galactosidase is an enzyme

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Classes of Proteins

• Transcription Factors
• Control expression of genes
• Hormones
• Control body function
• Antibodies
• Fight infection
• Enzymes
• Speed up chemical reactions
• Carrier molecules
• Hemoglobin -Carries oxygen in the blood

• Structural
• Collagen
• Found in bone and skin
• Keratin
• Makes hair and nails
• Fibrin
• Helps clot blood
• Elastin
• Major part of ligaments

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Proteins AreDiverse In Structure

• Proteins can do many things because they are
  structurally diverse
• Differ in many properties
• Size
• Shape
• Charge distribution
• Hydrophobicity
• Solubility properties

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Variability ComesFrom Amino Acids

• 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 gtgt bigger - from 100s to 1000s
• Allows for great diversity

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Amino Acids

• All amino acids have a carboxyl group and an
  amino group
• A different R group is attached to each amino
  acid

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Amino Acid
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• R groups make each amino acid different
• Some are
• Polar
• Nonpolar
• Charged
• Acids
• Bases

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Twenty Amino acids
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Folding

• 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

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Protein StructureAnd Function
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Shape Is Critical

• Change in one amino acid can change the structure
  of the protein with a large effect on function
• Sickle cell anemia

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Sickle Cell Anemia

• 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 fromMedline Plus
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Levels of ProteinStructure

• Protein structure is complex and important, so it
  is classified into
• 1 - Primary
• 2- Secondary
• 3 -Tertiary
• 4- Quaternary

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Primary Structure

• Linear sequence of amino acids
• Peptide bond (covalent bond) holds it together
• Beads on a string

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Primary Structure
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Peptide Bonds

• R O H R
• l ll l
  l
• NH2 C C 0 H H N CCOOH
• l
  l
• H H
• ?
• R O H R
• l ll l l
• NH2 -- C C N C COOH H2O
• l l
• H H

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Secondary Structure

• Regular repeating patterns of twists or kinks of
  the amino acid chain
• Examples
• Alpha helix
• Beta pleated sheet

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Secondary Structure

• 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)

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Figure from National Human Genome Research
Institute, by artist Darryl Leja. Used with
permission. 
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TertiaryStructure 3

• 3-D Globular Configuration formed by bending and
  twisting of the polypeptide chain
• Stabilized by
• Hydrogen bonds
• Electrostatic interactions
• (Positive and negative)
• Hydrophobic interactions
• Sometimes covalent bonds
• Disulfide bonds

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QuaternaryStructure 4

• Two or more polypeptide chains associate with
  each other

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ß-Galactosidase

• Link to Protein Data Bank for picture of
  molecular image of ß-galactosidase
• www.pdb.org

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Higher OrderStructure

• Higher order (secondary, tertiary, quaternary)
  structure is relatively weak
• In nature, weakness of noncovalent interactions
  is important
• Flexibility
• Enzymes change shape when bind their substrates
• Necessary for proper function

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How Proteins Lose Normal Structure And Function

• Primary structure hard to disrupt covalent bonds
  are strong

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How Proteins Lose Normal Structure And Function

• 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

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How Proteins Lose Their Structure And Function

• 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

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Higher OrderStructure In Lab

• 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

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Denaturation
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ManipulatingHigher Order Structure

• 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

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Analyzing Protein Structure

• 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

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X-rayCrystallography

• Isolate and purify protein
• Form a crystal of the protein
• Molecules of the protein are arranged in an
  orderly lattice
• Dissolve protein in solvent
• Precipitate into a crystal
• X-Ray the crystal

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X-rayCrystallography

• Analyze diffraction pattern using software
• Make electron density map
• Process used to take years
• Different versions of crystal for comparison
• Each with different heavy metal in lattice to
  provide reference point
• New X-ray sources - synchrotrons have reduced
  data collection time to few days

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X-rayCrystallography

• Synchotron - Argonne National Lab
• Still takes weeks to go from gene sequence to 3-D
  structure
• (mrsec website, nanotechnology at UW-Madison)

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StructuralGenomics

• 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

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Nuclear MagneticResonance

• Similar to MRI - Magnetic Resonance imaging in
  medicine
• No need for crystals, proteins in solution
• Works for relatively simple proteins