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Chapter 5:
Protein Function –Binding
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
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
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
Hemoglobin and Oxygen/CO2 Binding
Structures of Porphyrin and Heme
Coordination Positions – Bind with His93 = His F8
Myoglobin
Ligand Binding
θ = Fraction of Protein’s Ligand Binding Sites Bound to Ligand
Oxygen Binding to Myoglobin
What are the Kd’s ?
A EOC Problem 1: Relates Kd with “affinity” for ligand
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 oxymyoglobin.
What about proteins without a chromophore? and binding
colorless ligands?
Equilibrium dialysis
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.
Carbon Monoxide Binds Heme better than Oxygen
Due to Steric Effect of His E7 (see next slide)
Comparison of Myoglobin and a Hemoglobin
Polypeptide
Amino Acid Sequence of Myoglobin and Hemoglobin
Polypeptides
Grey Conserved, Pink Conserved in all known Hemoglobins
Hemoglobin Structure Showing Interchain
Contact Points
Contact Points in Primary Structure
Hemoglobin has Two Different Folded States
TR
Oxygen Binding Curves
EOC Problem 6
gets you further
into cooperativity
in oxygen
binding.
Knowing this will
help in Class.
Cooperative Binding
Hill Plot
CO Binds Well
Increased
Exposure to
even Low
Levels of CO
results in
COHb!
+
The effect of
exercise is
trivial.
This is Fatal
Two Models of Sigmoid Curves
Monod-Wyman-Changeux
Koshland (Induced Fit)
Both Models Fit the Data
Hb Oxygen Carrying Ability: the Bohr Effect
CO2 Transport on N-terminal Amino Groups
Effect of Altitude and 2,3 bisphosphoglycerate
EOC Problem 3:
examines this
phenomena...be sure
you know this for
Class.
BPG Fits into the Hole of the Doughnut
R state without
and with BPG
Positively charged R
groups are in blue.
Normal RBC vs Sickle Cell RBC’s
The Difference In Shape is due to One
Amino Acid Change Glu6  Val6 in Beta
Separation of Protein
Fragments
The Classic Paper
Chromatography +
Electrophoresis
Image from
Chapter 3
Hb-S Polymerizes in
the Deoxy form
Lymphocytes Have Binding Proteins
A
Antibodies have at least 2 Antigen Binding Sites
IgG
Carbohydrate Bound
Here
IgG Protein Structure and Binding an Antigen
IgM Has 10 Ag Binding
Sites
IgG and IgM are the
major circulating
antibodies
Cartoon of an ELISA
ELISA to Detect Herpes Simplex Virus in Blood
Samples
Positive
Negative
Controls
Antibodies Used with PAGE: Immunoblots
Binding receptors initiates Phagocytosis.
A
Myosin
Myosin Aggregate
Actin Filament
Myosin Contacting an Actin Filament
Muscle Structure
Electron Microscopy of Relaxed and Contracted
Muscle
Myosin-Actin Model
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 Hb’s 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.