PROTEIN FUNCTION

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 Functions of Globular Proteins
➢ Storage of ions and molecules
✓ Myoglobin Ferritina
✓ Ferritin

➢ Transport of ions and molecules

✓ Hemoglobin Hemoglobina
✓ Serotonin transporter

➢ Defense against pathogens

✓ Antibodies Interleucina
✓ Cytokines

➢ Muscle contraction
✓ Actin Actina
✓ Myosin

➢ Biological catalysis
✓ Chymotrypsin Lisozima
✓ Lysozyme 2
 Protein function
I/ Interaction with ligands

➢ Oxygen storage and transport: myoglobin and hemoglobin as

examples of protein evolution.

✓ Allosterism and cooperativity of hemoglobin.

✓ Different forms of hemoglobin: physiological adaptation

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 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

➢ The region in the protein where the ligand binds is called the binding site.

➢ Ligand binds via same noncovalent interactions that dictate protein structure
✓ Allows the interactions to be transient

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

ka
+
kd
P L PL
➢ This interaction can be described quantitatively by the association
rate constant ka or the dissociation rate constant kd.

➢ After some time, the process will reach the equilibrium

k a [P]  [L] = k d [PL]
where the association and dissociation rates are equal.

➢ The equilibrium composition is characterized

✓ by the equilibrium association constant Ka [PL] 1

Ka = =
[P]  [L] Kd
✓ or the equilibrium dissociation constant, Kd.

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 Binding: Analysis in Terms of the Bound Fraction

[PL]
=
[PL] + [P] K a [L][P] =
[ L]
= 1
[PL] 1 K a [L][P] + [P] [ L] +
Ka = = Ka
[P]  [L] Kd
[PL] 1 [PL]
Ka = K a==
[P]  [L] K[P d ]  [ L]

[ L]
=
[ L] + K d

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 100% de saturación
% saturación

50% de saturación

Ligando libre

Ligando unido a la proteína

kd [L]
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 Binding: Graphical Analysis

➢ The fraction of bound sites depends on the free ligand concentration and Kd.
The same as 100%

➢ Experimentally:
✓ ligand concentration is known

✓ Kd can be determined graphically

✓ The [L] at which half of the available

ligand-binding sites are occupied is
equivalent to 1/Ka, or Kd.

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 Reversible binding of a protein to Oxygen

➢ Structure of Myoglobin and Hemoglobin

✓ Myoglobin

Myoglobin. (PDB ID 1MBO): The heme is bound in a pocket made

up largely of the E and F helices, although amino acid residues from
other segments of the protein also participate.

Two coordination bonds to Fe2+ that are perpendicular to the

porphyrin ring system.
▪ One is occupied by a His residue, the proximal His
▪ the other is the binding site for oxygen. The remaining four
coordination bonds are in the plane of, and bonded to, the flat
porphyrin ring system

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 Grupo hemo

Mioglobina Hemoglobina
α1 β2

β1 α2
1 subunidad 4 subunidades

α :144 aminoácidos
153 aminoácidos
β :146 aminoácidos
1 grupo hemo 4 grupos hemo 10
11
 Hemoglobin Binds Oxygen Cooperatively
➢ Hemoglobin (Hb) is a tetramer of two subunits (a2b2).
➢ Each subunit is similar to myoglobin.
➢ http://biomodel.uah.es/model1j/prot/hb-oxidesoxi.htm

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 Oxygen Binding to Myoglobin

➢ When a ligand is a gas, binding is expressed in terms of

partial pressures.

pO 2
=
[L] =
K d + [L] p50 + pO 2

P50=0,26 kPa = 1,95 mmHg

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% Sat
100

80

60

40

20 P50 = 1,95
P50 = 30

pO2
0 (mmHg) 14
0 20 40 60 80 100
100 98
97
91

80

60 60

40
32
Tejido en ejercicio

Tejido en reposo

20

Pulmón
0
0 20 40 60 80 100 15
pO2 (mmHg)
 Tejido en Tejido en
Pulmón 100 mmHg 30 mmHg 20 mmHg
reposo ejercicio

1. Si consideramos a la míoglobina como

transportadora de O2
Tejido en O2 liberado en el tejido
Pulmón
reposo 98% - 97% = 1%
98%
97%
Tejido en
Pulmón O2 liberado en el tejido
ejercicio
98% 98% - 91% = 7%
91%

2. Si consideramos a la hemoglobina como

transportadora de O2
Tejido en
Pulmón O2 liberado en el tejido
reposo
98% 98% - 60% = 38%
60%
Pulmón Tejido en
O2 liberado en el tejido
ejercicio
98% 98% - 32% = 66%
32%

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 Mioglobina Hemoglobina

1 subunidad 1 grupo hemo 4 subunidades 4 grupos hemo

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 How Can Affinity to Oxygen Change?
➢ It 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
▪ recognized by sigmoidal binding curves
✓ Negative cooperativity
▪ first binding event reduces affinity at remaining sites

Subunit Interactions in Hemoglobin

➢ The strongest subunit interactions (highlighted)

occur between unlike subunits.
➢ When oxygen binds, the α1β1 contact changes little,
but there is a large change at the α1β2 contact, with
several ion pairs broken.

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 R and T States of Hemoglobin

T = tense state R = relaxed state

✓ more interactions, more stable ✓ fewer Interactions, more flexible
✓ lower affinity for O2 ✓ higher affinity for O2
For Effective Transport, Affinity Must Vary with pO2
➢ O2 binding triggers a T → R conformational change.
➢ Conformational change from the T state to the R state involves
breaking ion pairs between the α1-b2 interface.
A sigmoid binding curve can be viewed as a hybrid curve reflecting a
transition from a low-affinity to a high-affinity state. Because of its
cooperative binding, as manifested by a sigmoid binding curve,
hemoglobin is more sensitive to the small differences in O2 concentration
between the tissues and the lungs, allowing it to bind oxygen in the lungs
(where pO2 is high) and release it in the tissues (where pO2 is low). 19
 pH Effect on O2 Binding to Hemoglobin

➢ The pH of blood is 7.6 in the lungs

➢ and 7.2 in the tissues.
➢ Experimental measurements on hemoglobin binding
are often performed at pH 7.4.
➢ Does acidity increase or decrease the Kd?

➢ Actively metabolizing tissues generate H+, lowering the pH

of the blood near the tissues relative to the lungs
(catalyzed by carbonic anhydrase).

CO2 + H2O HCO3− + H+

➢Hb Affinity for oxygen depends on the pH.

✓ H+ binds to Hb and stabilizes the T state.
✓ leads to the release of O2 (in the tissues)

➢ The pH difference between lungs and metabolic tissues

increases efficiency of the O2 transport.
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➢ This is known as the Bohr effect.
 Hemoglobin and CO2 Export
➢ CO2 is produced by metabolism in tissues and must be exported.
➢ 15–20% of CO2 is exported in the form of a carbamate on the amino terminal
residues of each of the polypeptide subunits.

➢ Notice:
o The formation of a carbamate yields a proton that can contribute to the Bohr effect.
o The carbamate forms additional salt bridges, stabilizing the T state.

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2,3-Bisphosphoglycerate Regulates O2 Binding

➢ Negative heterotropic regulator of Hb function

➢ Present at mM concentrations in erythrocytes
✓ produced from an intermediate in glycolysis
➢ Small negatively charged molecule,
➢ binds to the positively charged central cavity of Hb
➢ Stabilizes the T state

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% Sat En el Tejido En el pulmón a 4500 m En el pulmón a nivel del mar
100
98

90

80 38%
30%

60

40

20

pO2
0 (mmHg) 23
0 20 40 60 80 100
% Sat En el Tejido En el pulmón a 4500 m En el pulmón a nivel del mar
100
98

85
80

37%
60

48
40

20

pO2
0 (mmHg) 24
0 20 40 60 80 100
 pO2 en pulmón pO2 en pulmón
pO2 en tejido a 4500 m a nivel del mar

100
98

90
85

60

50
48

0,0
pO2 (en kPa) 25
30 mmHg
 2,3-BPG Allows for O2 Release in the Tissues
and Adaptation to Changes in Altitude

➢ The BPG concentration in normal human

blood is about 5 mM at sea level and about
8 mM at high altitudes.
➢ At sea level, hemoglobin is nearly saturated
with O2 in the lungs, but just over 60%
saturated in the tissues, so the amount of
O2 released in the tissues is about 38% of
the maximum that can be carried in the
blood.
➢ At high altitudes, O2 delivery declines by
about one-fourth, to 30% of maximum.
➢ An increase in BPG concentration,
however, decreases the affinity of
hemoglobin for O2, so approximately 37%
of what can be carried is again delivered to
the tissues.

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 Summary

➢ how ligand binding can affect protein function

➢ how myoglobin stores oxygen
➢ how hemoglobin transports O2, protons, and CO2

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