Protein Function2014

Protein function

• Chapter 5.1
• Myoglobin: structure, O2-binding
• Hemoglobin: structure, cooperativity in O2binding, Hill constant, allosteric interactions, Bohr Effect, BPG-binding and effect • Abnormal Hemoglobins

Functions of Proteins
Fibrous proteins: collagen, keratin, silk - give tensile strength, shelter, protection
Globular proteins:
• Storage of ions and molecules
– myoglobin, ferritin
• Transport of ions and molecules
– hemoglobin, serotonin transporter
• Defense against pathogens
– antibodies, cytokines
• Molecular motors
– myosin, kinesin
• Biological catalysis
– chymotrypsin, lysozyme
– Many more

Interaction with Other Molecules

• Reversible, transient process of chemical equilibrium:


A + B AB A + B
• A molecule that binds 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 non-covalent forces, which enables the interactions to be transient

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Specificity: Lock-and-Key Model
• Proteins typically have high specificity: only certain ligands bind
• High specificity can be explained by the complementarity of the binding site and the ligand.
• Complementarity in
–size,
–shape,
–charge,
–or hydrophobic / hydrophilic character
• “Lock and Key” model by Emil Fisher (1894) assumes that complementary surfaces are preformed.

+

Specificity: Induced Fit
• Conformational changes may occur upon ligand binding
(Daniel Koshland in 1958).
– This adaptation is called the induced fit.
– Induced fit allows for tighter binding of the ligand
– Induced fit can increase/change the affinity of the protein for a second ligand
• Both the ligand and the protein can change their conformations

+

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Myoglobin: Function
• Metabolism is much more efficient when under aerobic conditions
• Glycolysis of 1glucose results in 2 ATP.
• When under oxidative conditions, glycolysis is followed by citric acid cycle and oxidative phosphorylation: results in 30 or 32 ATP
• Need for OXYGEN
• Problem: Oxygen is not very soluble in water
(only 10-4 M)
• Myoglobin is major O2-storage and transport protein in muscle.

Function of Myoglobin
• Need to store oxygen for metabolism
• Protein side-chains lack affinity for O2
• Some transition metals bind O2 well but would generate free radicals if free in solution
• Organometallic compounds such as heme are more suitable but Fe2+ in free heme could be oxidized to Fe3+ if two heme molecules share an oxygen molecule
• Solution:
– Capture the oxygen molecule with heme that is sequestered inside the protein
– In mammals, myoglobin is the main oxygen storage protein 3

Structure of heme

• Fe is coordinated by porphyrin N-atoms.
• The porphyrin system is fully conjugated, averaging the distances NFe
• Heme side chains are: 4 methyl, 2 vinyl, 2 propionate

Myoglobin: Structure

• 8 a-helices (A-H) in a globular arrangement
• A single heme group is positioned in hydrophobic pocket between helices E and F.
• Heme: porphyrin derivative that complexes Fe(II) with 4
N-atoms of porphyrin system, one coordination site is bound to protein-His, the 6th coordination binds O2.
• If isolated heme binds O2, Fe (II) is irreversibly oxidized to
Fe(III), which does not bind O2. This oxidation is prevented in Mb by binding of heme to globin.

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Binding of Carbon Monoxide
• CO has similar size and shape to O2; it can fit to the same binding site
• CO binds over 20,000 times better than O2 because the carbon in
CO has a filled lone electron pair that can be donated to vacant dorbitals on the Fe2+
• Protein pocket decreases affinity for CO, but is still binds about
250 times better than oxygen
• CO is highly toxic as it competes with oxygen. It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation

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Binding: Quantitative Description
• Consider a process in which a ligand (L) binds reversibly to a site in the protein (P)

ka
P

+

L

kb

PL

• The kinetics of such a process is described by: the association rate constant ka the dissociation rate constant kd
• After some time, the process will reach the equilibrium where the association and dissociation rates are equal

k a [P] [L]  k d [PL]

• The equilibrium composition is characterized by the the equilibrium constant Ka

Ka 

[PL] k  a
[P]  [L] kd

Binding: Analysis in Terms of the Bound Fraction
• In practice, we can often determine the fraction of occupied binding sites



[PL]
[ PL]  [ P]

• Substituting [PL] with Ka[L][P], we’ll eliminate [PL]



K a[L][P]
K a [L][P ]  [P]

• Eliminating [P] and rearranging gives the result in terms of equilibrium association constant:
• In terms of the more commonly used equilibrium dissociation constant:



[L ]
[L ] 



1
Ka

[ L]
[ L]  K d

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pO2 in arterial blood ~ 13 kPa
Q = 0.97 = fractional occupation of Mb with O2 pO2 in venous blood ~ 4 kPa
Q = 0.91
P50: 0.13-0.26 kPa = ~1-2Torr
Under physiological O2-pressure
Mb is almost fully oxygenized

Could Myoglobin Work as Good
O2 Transporter?
• pO2 in lungs is about 13 kPa: it sure binds oxygen well
• pO2 in tissues is about 4 kPa: it will not release it!
• Would lowering the affinity (P50) of myoglobin to oxygen help?

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For Effective Transport Affinity
Must Vary with pO2
• pO2 in lungs is about 13 kPa: it sure binds oxygen well • pO2 in tissues is about 4 kPa: it releases about half of it at pH 7.6

How Can Affinity to Oxygen
Change Like This?
• Must be a protein with multiple binding sites
• Binding sites must be able to interact with each other
• This phenomenon is called cooperativity
– positive cooperativity: first binding event increases affinity at remaining sites
– negative cooperativity: first binding event reduces affinity at remaining sites
Positive cooperativity can be recognized by sigmoidal binding curves 8

Hemoglobin: oxygen transport in blood
Hb: a highly regulated, sophisticated O2-delivery system: a2  2 stoichiometry
Other members of the globin protein family: a-like:  in 22 (early embryogenesis)
-like : ,  in a2 2 (in fetal Hb ),
 (in primates)

Mb and Hb monomer are very similar in structure, but only 18 % identical aa

O2-binding changes the conformation of the Hbtetramer
Upon oxygenation the a1 1 and a2 2 dimer interfaces rotate relative to each other and shift closer, affecting certain ion pairs.

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Binding of O2 to Hb in one binding site results in conformational change in all Hb subunits: allosteric effect causes cooperativity of binding sites

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Cooperative oxygen binding to Hb
Sigmoidal curve indicates cooperative binding. Upon binding of 1st O2 the affinity for additional O2-binding increases. P50 of Hb in whole blood is 2.6 kPa, P50 of
Mb is 0.26 kPa.

Hill-plot of oxygen binding to Hb
The maximal slope is defined as n (Hill constant): n>1: positive cooperativity n=1: no cooperativity n<1: negative cooperativity

Low O2-affinity

High O2-affinity

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Low O2-affinity

High O2-affinity

R and T States of Hemoglobin
• T = Tense state, O2 binding triggers a T conformational change

R

• R = Relaxed state, this state has higher affinity for O2 than the T state
• Deoxyhemoglobin subunits are more stable in the T state • Conformational change from the T state to the R state involves breaking ion pairs between the a1-2 interface Mechanisms of cooperativity
Sequential model by Koshland, Nemethy, Filmer (KNF)

Symmetry model by Monod, Wyman and Changeux (MWC)
High O2 affinity

Low O2 affinity

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pH Effect on O2 binding to
Hemoglobin
• Blood in lungs has higher pH than blood in capillaries of metabolic tissues
• Affinity for oxygen depends on the pH
– Oxygen binds well at higher pH
– Oxygen is released well at lower pH

• The pH difference between lungs and metabolic tissues increases the O2 transfer efficiency
• This is known as the Bohr effect

Bohr Effect (Christian Bohr, 1904)

 Increase in pH increases O2 affinity.  Decreasing pH leads to release of O2.

Chemical basis for Bohr effect: formation of salt bridges in T-Hb depends on protonation.
Protonation stabilizes Hb in T form

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Hemoglobin and CO2 Export
• CO2 is produced by metabolism in tissues and must be exported
• Some CO2 exported as dissolved bicarbonate in the blood, CO2 reacts to HCO3- and H+ (catalyzed by carbonic anhydrase in erythrocytes). • Some CO2 is exported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits.
• Carbamate formation stabilizes T-state, causes O2 release. The formation of a carbamate also yields a proton (see next slide) which can bind to hemoglobin and promotes oxygen dissociation The released H+ binds to His to stabilize T state, O2 release

Bohr effect on equilibrium
In lungs: high pO2 stabilizes
R state: H+ released, causes CO2 to be released from Hb, decreased pH also causes CO2 to bubble out of solution, CO2 exhaled!
In metabolically active tissue: protonation and carbamate formation stabilize T-state:
O2 release to where it is needed!

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In muscle with high turnover, lactate-formation upon reduction of pyruvate causes release of oxygen.

Summary pH effects:
Increase in [H+] (pH ↓) leads to O2 release by stabilizing
T-form of Hb.
In lungs, high pO2 leads to binding of O2, this stabilizes
R state and H+ release, increase in [H+] forces CO2 out.

Effect of BPG on oxygen binding to Hb

YO2 = Q

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2,3-Bisphosphoglycerate modulates oxygen binding to Hb

2,3-BPG binds in central cavity of Hb in Tform decreasing affinity for oxygen

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Mechanisms of cooperativity
Symmetry model by Monod, Wyman and Changeux

Bisphosphogycerate stabilizes T-state: low O2 affinity

Adaptation to different pO2 pressure possible Fetal Hb (a22) binds 2,3-BPG with less affinity, oxygen transfer from maternal blood to fetal blood possible

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Bilirubin in Heme degradation

Abnormal hemoglobins:
• Mutations that destabilize tertiary and quaternary structure: alter O2-binding affinity and Hill constant.
• Unstable Hb are degraded: hemolytic anemia.
• Mutations that allow Fe (II) to Fe (III) oxidation.
• Mutations causing too high O2-affinity, not enough release possible. • Thalassemia: imbalanced production of globins (genetic, autosomal recessive, prevalent among Mediterranean peoples, also 18 % carriers in Maldives, Asia)
• Single amino acid mutation (Val instead of Glu) causes sickle cell anemia (genetic, 10 % African American, 25 %
African carriers)

Sickle cell anemia

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Single amino acid exchange causes polymerization of Hb,
Val replaces Glu

Comparison of normal and sickle cell erythrocytes The pain in his joints throbbed in and out, with every beat of his heart. Pain, like a million tiny needles jabbing in and pulling out, with every beat of the heart.
“Mommy! Make it stop! Make it stop - now.” He needed to calm himself in the midst of pain and panic. He had tried rhythmic breathing. He tried thinking of pleasant things. He tried focusing on the touch of his mother’s hand.
After forcing fluids and keeping perfectly still, like a slowly receding tide, the waves of pain steadily washed away. Soon, he would be back to a weakened but more normal self.
From the web-site of Dr. Betty Pace, UTD http://www.utdallas.edu/biology/faculty/profiles/ pace.html

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