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.