protein structure and function. proteins are where it’s at proteomics gene regulation drug...
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Proteins are Where It’s Been Enzymes ß-galactosidase Antibodies Anti-Hepatitis B Hormones Human Growth Hormone Estrogen TestosteroneTRANSCRIPT
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PROTEIN STRUCTURE AND FUNCTION
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Proteins Are Where It’s At
• Proteomics• Gene regulation• Drug Discovery• Understanding evolution• Etc.
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Proteins are Where It’s Been
• Enzymes ß-galactosidase
• Antibodies Anti-Hepatitis B
• Hormones Human Growth Hormone Estrogen Testosterone
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Proteins are Where It’s Been
• Structural proteins Collagen
• Transportation Haemoglobin
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Proteins Are Us
• 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
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Transcription Factors Control expression of
genes Hormones
Control body function Antibodies
Fight infection Enzymes
Speed up chemical reactions
Carrier molecules Haemoglobin -carries
oxygen in the blood
Classes of Proteins• 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 >> 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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Image fromMedline Plus
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.
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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 lNH2 –C – C – 0 –H + H –N – C—COOH 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
1. (Positive and negative) Hydrophobic interactions Sometimes covalent bonds
1. 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
Flexibility1. Enzymes change shape when binding to
their substrates2. 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 lattice1. Dissolve protein in solvent2. 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
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
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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