Protein Function

1
Protein Function Binding
Chapter 5
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Function of Globular Proteins Ligand Binding
Learning Goals

• 1. Reversible binding of ligands is essential
• Specificity of ligands and binding sites
• Ligand binding is often coupled to conformational
  changes, sometimes quite dramatic (Induced Fit)
• In multisubunit proteins, conformational changes
  in one subunit can affect the others
  (Cooperativity)
• Interactions can be regulated
• 2. Illustrated by
• Hemoglobin, antibodies, and muscle contraction

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Functions of Globular Proteins

• Storage of ions and molecules
• myoglobin, ferritin
• Transport of ions and molecules
• hemoglobin, serotonin transporter
• Defense against pathogens
• antibodies, cytokines
• Muscle contraction
• actin, myosin
• Biological catalysis
• chymotrypsin, lysozyme

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Protein Interaction with Other Molecules

• Reversible, transient process of chemical
  equilibrium
• A B ?? AB
• A molecule that binds to a protein is called a
  ligand
• Typically a small molecule
• A region in the protein where the ligand binds is
  called the binding site
• Ligand binds via same noncovalent forces that
  dictate protein structure (see Chapter 4)
• Allows the interactions to be transient

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Hemoglobin and Oxygen/CO2 Binding
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Structures of Porphyrin and Heme
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Coordination Positions Bind with His93 His F8
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Myoglobin
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Ligand Binding
? Fraction of Proteins Ligand Binding Sites
Bound to Ligand
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Oxygen Binding to Myoglobin
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What are the Kds ?
A
EOC Problem 1 Relates Kd with affinity for
ligand
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How is the Binding Experiment Done?
With proteins such as Hemoglobin and Myoglobin,
the absorbance spectrum changes between free and
bound protein can be measured in a
spectrophotometer. The spectrum of
deoxy-myoglobin is different than
oxy-myoglobin. What about proteins without a
chromophore? and binding colorless
ligands? Equilibrium dialysis
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Human serum transferrin binds iron at pH 7.2 with
a Kd of 10-19 to 10-20 M EOC Problem 5 uses
simple inspection of the data to get Kd ! Make
sure you get this done before Class.
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Carbon Monoxide Binds Heme better than Oxygen Due
to Steric Effect of His E7 (see next slide)
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Comparison of Myoglobin and a Hemoglobin
Polypeptide
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Amino Acid Sequence of Myoglobin and Hemoglobin
Polypeptides
Grey Conserved, Pink Conserved in all known
Hemoglobins
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Hemoglobin Structure Showing Interchain Contact
Points
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Contact Points in Primary Structure
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Hemoglobin has Two Different Folded States
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T ? R
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Oxygen Binding Curves
EOC Problem 6 gets you further into cooperativity
in oxygen binding. Knowing this will help in
Class.
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Cooperative Binding
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Hill Plot
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CO Binds Well
Increased Exposure to even Low Levels of CO
results in COHb! The effect of exercise is
trivial.
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This is Fatal
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Two Models of Sigmoid Curves
Monod-Wyman-Changeux Koshland (Induced
Fit)
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Both Models Fit the Data
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Hb Oxygen Carrying Ability the Bohr Effect
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CO2 Transport on N-terminal Amino Groups
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Effect of Altitude and 2,3 bisphosphoglycerate
EOC Problem 3 examines this phenomena...be sure
you know this for Class.
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BPG Fits into the Hole of the Doughnut
R state without and with BPG
Positively charged R groups are in blue.
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Normal RBC vs Sickle Cell RBCs
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The Difference In Shape is due to One Amino Acid
Change Glu6 ? Val6 in Beta
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Separation of Protein FragmentsThe Classic Paper
Chromatography Electrophoresis
Image from Chapter 3
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Hb-S Polymerizes in the Deoxy form
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Lymphocytes Have Binding Proteins
A
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Antibodies have at least 2 Antigen Binding Sites
IgG
Carbohydrate Bound Here
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IgG Protein Structure and Binding an Antigen
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IgM Has 10 Ag Binding Sites
IgG and IgM are the major circulating antibodies
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Cartoon of an ELISA
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ELISA to Detect Herpes Simplex Virus in Blood
Samples
Positive Negative Controls
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Antibodies Used with PAGE Immunoblots
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Binding receptors initiates Phagocytosis.
A
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Myosin
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Myosin Aggregate
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Actin Filament
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Myosin Contacting an Actin Filament
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Muscle Structure
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Electron Microscopy of Relaxed and Contracted
Muscle
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Myosin-Actin Model
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Things to Know and Do Before Class

1. Know how binding studies are done and the meaning
   of Kd.
2. Know how conformation of a protein affect ligand
   binding (models of myoglobin and hemoglobin)for
   loading and off-loading oxygen.
3. How altitude affects Hbs oxygen binding.
4. Why Hb-S causes red blood cells to change shape
   and what affect that has on Hb-S individuals.
5. How antibodies bind to antigens.
6. Be able to do EOC Problems 1,3, 5-7.