David L. Nelson and Michael M.

Cox

Lehninger Principles of
Biochemistry
Fourth Edition

Chapter 5:
Protein Function

Copyright © 2004 by W. H. Freeman & Company

[1] Protein Functions
Transport –
myoglobin transports and stores oxygen in muscle,
hemoglobin transports oxygen in the blood (also CO2).

Organized Movement - actin & myosin - movement

tropomyosin & troponin - control

Immune response - antibodies, produced by B lymphocytes,

recognize foreign macromolecules (antigens), for example
viruses!
Enzymes - carry out various chemical reactions

Receptors - bind to specific molecules (ligands, messengers)

and allow signals from the outside of the cell to be
transferred to the inside of the cell.
An example is a G-coupled protein receptor such as the
human opioid receptor that is involved in pain/ analgesia,
emotions and addiction.

Opioid Receptors bind to

morphine (analgesia),
heroin (addiction),
enkephalin, endorphin
that cause various effects
in the body …
Figure 12-12, p. 436
[2] A few concepts in protein functions

① Reversible binding to a molecule (ligand).

② A ligand binds to the binding site of a protein, which is
structurally complementary to the ligand.  Specific
binding
③ Proteins are flexible (They breathe!)  can undergo
conformational change
Ligand induced conformational change  induced fit

Induced-Fit Model of Enzyme catalysis

[2] A few concepts in protein functions
④ Conformational change propagates. 
Conformational change of a protein (subunit) often affects
the conformational change of other interacting proteins
(subunits).
⑤ The protein-ligand interactions may be regulated.
[3] Reversible Binding of a Protein to a Ligand:
Oxygen-Binding Proteins

 Why do we need a transporter for O2?

 Low solubility of O2 (Table 2-3, 0.035 g/L at 50oC)
30-fold lower to CO2 (0.97 g/L)

 Why a large protein (myoglobin, 153 a.a., 17 kDa),

why not a small peptide?

 Why a tetramer (hemoglobin)?

① We need an O2 carrier for storage/transportation.
 Reversible binding, No reaction w/ O2.
A compound with an adequate affinity to O2
 Biomolecules including a. a. or sugars do not bind O2 well.
 Fe2+ (Cu2+)

② Handle with care. (High reactivity of O2)

 O2 is a diradical.
16O – 1s22s22p4

2s2 2p4
 Eager to accept electron(up to 2).
 Easily reduced. Oxygen is a good oxidant.
(½O2 + 2 e‒ + 2 H+  H2O)
Oxidation : the process whereby a molecular species
loses an electron.
Reduction : the process whereby an electron is gained.
② Handle with care. (High reactivity of O2)
O2 is a diradical.
O O
Oxygen molecule : O=O

Molecular orbital of O2 molecule:

2p*

x* y*

x y

2p
Reactive Oxygen Species
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/ROS.html
 Due to its high reactivity, a number of highly reactive
free radicals are generated from oxygen in living systems
as an unavoidable consequence of aerobic respiration.
 O2‒ (superoxide anion), HO (hydroxyl radical),
H2O2 (hydrogen peroxide).
 All these partially reduced species of oxygen are toxic.

 Free Fe2+ ions (that we need for transfer) participate in

radical formation reactions and are oxidized to Fe3+.
 Its reactivity needs to be restrained (controlled).
 Coordinated to 4 N atoms in heme and to a His side chain N
in protein (myoglobin !!), leaving one
“open” coordination bond to O2.
 [5] Protein Structure Affects How Ligands Bind

 To reduce unwanted binding of

heme to other ligands (CO), the
heme group is well sequestered
inside of the protein.
 The O2 moves in and out of the
binding pocket through
“transient cavities” produced by
the “breathing” of the protein,
such as the rotation of the distal
His side chain in 10-9 sec.
 [4] Reversible Protein-Ligand Interaction

P + L ⇄ PL
[P] [L]
Kd (dissociation constant) = (M)
[PL]
Fraction of occupied ligand binding sites,
binding sites occupied [PL]
 (theta) = total binding sites
=
[PL] + [P]
①

[P] [L]
[PL] = ②
Kd
Substituting [PL] in ① with ② and rearranging terms gives,
[L]
 = ③ lower Kd  higher affinity
[L] + Kd
 [L]
 =
[L] + Kd

 Hyperbolic.
 At high [L],
binding sites are saturated.
 When [L] = Kd,
 = 0.5 (half occupied).
 When [L] = 9 Kd,
 = 0.9 (90% occupied).
 Kd is the concentration of L needed to bind half of the binding site.
 The more tightly a protein binds to a ligand, the lower the [L]
required for half saturation.
 Lower Kd  higher affinity.
 Oxygen (O2) binding to myoglobin follows the same pattern.
Here, [L] = [O2], Kd = [O2]0.5
[O2] pO2
Then,  = =
[O2] + [O2]0.5 pO2 + P50
P50 - the partial pressure of oxygen at [O2]0.5

Note that the O2 binding to

myoglobin is relatively
insensitive to small change in
[O2] (pO2) when pO2 is larger
than certain value.
Myoglobin with its high affinity
for O2 is good for storage.
But not good for transport.
• So, we need a good transporter (a mailman)
that knows when to pick up and when to
deliver the parcel.
• A transporter with varying affinity to ligand.
[6] Hemoglobin (Hb), the transporter

 Found in erythrocytes (RBC, red blood cell),

~34% by weight.
 22 tetramer.
 Each subunit is
structurally similar to
myoglobin.

 Sensitive to small
changes in [O2]
thanks to the
quaternary structure.
 [7] Hemoglobin binds O2 Cooperatively 1 kPa
Transport problem:  0.01 atm
pO2 at the lungs (pick-up point) is ~ 13.3 kPa,
while in the tissues (release point) pO2 is ~ 4 kPa.
We need a transporter
whose affinity changes
from high at high pO2
(for easy pick-up)
to low at low pO2
(for easy deliver).
Sigmoidal
Positive cooperativity
O2 binding to individual subunits
of Hb can alter the affinity for
O2 in adjacent subunits.
⇒ Hemoglobin exists in two conformations,
the low-affinity T state, and the high-affinity R state.

Low affinity High affinity

⇒ The T state is more stable, and unbound Hb (deoxyHb)
exists predominantly in the T form.
⇒ When O2 binds to a Hb subunit in the T form, it triggers a
conformational change in adjacent subunits, converting
them to the R form.
⇒ a sigmoidal (positive coorperative) binding curve.

puckered planar
Allostery:
A phenomenon
whereby the binding of
a ligand (effector) to
one site changes
through the
conformational change
the binding properties
of another site on the
same protein.
[8] The Hill Equation and the Measure of Cooperativity
(1) Why cooperativity? (why Hb cannot be 4Mb?)
 Mb has high affinity for O2.
 It is saturated with O2 at the pO2 found in the lungs.
 It is nearly saturated at the pO2 found in the tissues.
 If Hb were like 4Mb, it would be saturated
with O2 in the lungs, but it would not
release much O2 in the tissues,
because its affinity would be equally high as Mb's.
 If Hb were like 4Mb, but with lower affinity
for O2, it could release O2 in the tissues,
but it would have difficulty loading O2
in the lungs, because of its low affinity.

 It is essential that Hb's saturation curve

for O2 binding be sigmoidal, rather than hyperbolic.
· In this case, Hb is saturated at pO2 found
in the lungs, but binds O2 weakly at pO2 found in the tissues.
 This is possible due to cooperativity.
⇒The binding of O2 at one site in Hb increases the
affinity for O2 at other sites in the same Hb molecule.
(2) Binding equation for Hb

For simplicity, we will consider the special case of infinite

cooperativity:
all O2 molecules bind at once.

Hb + 4 O2 ⇄ Hb(O2)4

For a protein with n biding sites,

P + nL ⇄ PLn

(Note that here we are assuming all or nothing binding with

no observable intermediates PL1, PL2, etc.
That is, infinite cooperativity is assumed.)
 P + nL ⇄ PLn
[P] [L]n
Kd =
[PLn]
[PLn]
= ①
[PLn] + [P]
[P][L]n
[PLn] = ②
Kd
[L]n
 = ③
[L]n + Kd


log = n log[L] ‒ log Kd, where Kd = [L]n0.5 ④
1‒
slope = n y-intercept = ‒ log Kd
 A plot of log [/(1 - )] vs log [L] (the Hill plot) has a slope
of n.
 In physically impossible all-or nothing binding (infinite
cooperativity), the slope n is equal to the number of the
binding sites, i.e. 4 for Hb.
In reality, it is never n, and is called nH, the Hill coefficient

For Hb, nH = 2.8-3.0

Hill coefficient, nH, reflects

the degree of interaction
(cooperativity) between
the binding sites.
Interpretating the Hill Plots
① nH = 1 ⇒ Ligand binds noncooperatively, e.g. Mb and O2.
This situation can happen even with a multisite protein if the
binding sites do not communicate with one another.

② 1 < nH < n ⇒ Positive cooperativity, e.g. Hb and O2

Typical for a cooperatively binding protein.
Ligand binding increases the affinity for further ligand binding.

③ nH = n ⇒ In this hypothetical situation the molecule is wholly

cooperative. In such a situation, one hemoglobin molecule would fill
up its four oxygen-binding sites before any others had taken oxygen,
so that only wholly unliganded and wholly liganded molecules would
be present at any point in the binding process.
④ 0 < nH < 1 ⇒ Negative cooperativity.
Ligand binding decreases the affinity for further ligand binding.
Rare in nature.
[9] Hemoglobin S and Sickel-Cell Anemia
A single mutation of GluVal at the position 6 of the  chain creates
a “sticky” patch on the surface.
Why the sickle-cell allele is common in certain (malaria infested)
parts of Africa?

The sickle cell traits (heterozygote, HbAHbS) red blood cells look
normal, but can sickle if the individuals become dehydrated or
suffer mild oxygen deprivation.

In high malaria areas of Africa:

Sickle cell amenia babies die from sickle cell.
Normal individuals risk dying from malaria infection.
Sickle cell trait individuals are immune from contracting the
malaria parasite (with unknown mechanism) and won’t die from
sickle cell.
[9] The Bohr Effect (p. 170-171)
CO2 produced by metabolic processes is hydrated to HCO3-

CO2 + H2O ⇄ H+ + HCO3-

Therefore, in tissues, pH ↓, [CO2] ↑.

The inverse effects of [H+] and
CO2 binding to Hb
on the binding affinity of Hb
for O2 helps Hb bind H+
and CO2 and release O2
in the tissues,
vice versa in the lungs.
[10] 2,3-Bisphophoglycerate (BPG) and high altitudes
(p. 171-1
BPG binds to Hb and regulates its affinity for O2.
At high altitudes (low pO2), blood [BPG] increases, and
the Hb affinity for O2 decreases in the tissues,
helping the release of O2 in the tissues.