Chapter 5:
Protein Function –Binding
Function of Globular Proteins: Ligand Binding
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
Learning Goals
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 ?
AEOC 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 oxy-myoglobin.
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
A
Binding receptors initiates Phagocytosis.
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.