Chapter 14 - Allostery

True/False and Multiple Choice (7)

1)

Which of the following can result in an allosteric modulation of activity:

a) covalent modification such as phosphorylation or acetylation
b) oligomerization
c) binding of a ligand
d) stabilizing an alternative conformation
e) all of the above

2)

The transcription of a gene is controlled by a transcription factor binding either

Glucose or Lactose. When there is 0.2mM of either Glucose or Lactose in the cell,
the gene is transcribed at about 10% of the maximum. When there more than 2mM
Glucose in the cell, the gene is fully induced. However, when there is 2mM Lactose
in the cell, there amount of transcription is approximately half of the maximum. At
40mM of either Glucose or Lactose in the cell, the gene is fully induced. The
transcriptional regulation is likely:
a) ultrasensitive with respect to both Glucose and Lactose
b) ultrasensitive with respect to Lactose but not Glucose
c) graded with respect to Glucose
d) ultrasensitive with respect to Glucose but not Lactose
e) hyperbolic with respect to Glucose but not Lactose

3)

In allosteric proteins, ligand binding can only result in positive cooperativity

True/False

4)

Which of the following is not a known allosteric effector of hemoglobin:

a) oxygen
b) bisphosphaoglycerate
c) low pH
d) myoglobin
e) CO2

5)

Bacterial chemotaxis involves only random, brownian movement

True/False

6)

7)

Because of the importance of the F-helix in oxygen binding, the allosteric

hemoglobin proteins of all organisms are tetrameric assemblies
True/False
Which of the following statements about the Hill coefficient are true:
i. it is the steepness of the log-log binding isotherm at the half saturation point
ii. allosteric systems have a Hill coefficient of exactly 1
iii.the maximum value for a dimeric protein is 2
a) only i
b) i and iii
c) ii and iii
d) all of the statements are true

Fill in the Blank (5)

1) When a system is ultrasensitive, the response to an input is _______ than expected
from the graded, hyperbolic response.
(sharper)
2)

Oxygen biases the equilibrium of hemoglobin towards the _____ state

(R or relaxed)

3)

The extent of flagellar clockwise rotation is governed by the

___________________ of the protein CheY
(phosphorylation)

4)

The __________ histidine residue of hemoglobin sense the effective size of the
heme iron atom
(proximal)

5)

An effect of co-localization is to greatly increase the local __________ of two

proteins
(concentration)

Long Answer/Essay (12)

1)

A dimeric glucokinase has a binding site for glucose in each subunit. The K D for
the first binding event is 1mM and the KD for the second event is 10uM. a) What is
the Hill coefficient? b) Is this protein positive or negatively cooperative with respect to
glucose binding?
Answer:
a)
nh = 2 / (1+(KD2/KD1))
= 2 / (1+(0.01/1))
= 1.8
b)
nh > 1, therefore it is positively cooperative

2)

A dimeric hemoglobin is isolated from a fish. Each subunit contains a binding

site for a Xenon gas molecule. The KD for the first binding event is measured at
23nM. The KD for the second binding event is measured at 3.5uM. a) What is the Hill
coefficient? b) Is this protein positive or negatively cooperative with respect to Xenon
binding?
Answer:
a)
nh = 2 / (1+(KD2/KD1))
= 2 / (1+(3.5/0.023))
= 0.15
b)
nh < 1, therefore it is negatively cooperative

3)

A dimeric enzyme with two identical binding sites has a Hill coefficent of 1.3. If
the KD of the first binding site is 200nM, what is the KD of the second binding site?
Answer:
KD2 = KD1x(2/nh - 1)2
= 200x(2/1.3-1) 2
= 58 nM

4)

What is the ratio of bound to unbound receptor (f/(1-f)) for a dimeric protein with
two binding sites (KD1 = 20nM, KD2 = 2 nM) at 15nM concentration of ligand?
Answer:
f/(1-f) = ([L]/KD1 + ([L]/KD1)* ([L]/KD2))/(1+[L]/KD1)
= (15/20 + (15/20)* (15/2))/(1+15/20)
= 3.64

5)

A dimeric allosteric protein is isolated. A scientist measures the K D1 for the first
binding site at 25nM and the Hill coefficient as 1.6. a) What fraction of the protein is
bound at 10nM concentration of ligand? b) A single point mutation abolishes all
cooperativity in the protein such that the protein binds its ligand with an apparent K D
equal to 25nM. What is the fraction of the protein is bound at 10nM concentration of
ligand?
Answer:
a)
KD2 = KD1x(2/nh - 1)2
= 25x(2/1.9 - 1)2
= 1.56 nM
f/(1-f) = ([L]/KD1 + ([L]/KD1)* ([L]/KD2))/(1+[L]/KD1)
= (10/25 + (10/25)* (10/1.56))/(1+10/25)
= 2.12
f = 2.12 - 2.12f
3.12f = 2.12
f = 2.12/3.12
f = .68
b)
f/(1-f) = [L]/KD
= 10/25
= 0.4
f = 0.4/1.4
= 0.29

6)

The first and second binding sites of a positively cooperative allosteric dimeric
protein have KDs of 100mM and 10uM respectively. a) Sketch the binding isotherms
of log(f/(1-f)) vs log([L]) b) What is the Hill coefficient?

Answer:
a)

b)
nh = 2 / (1+(KD2/KD1))
= 2 / (1+(10-5/10-1))
= 1.98
7)

The first and second binding sites of a negatively cooperative allosteric dimeric
protein have KDs of 100uM and 10mM respectively. a) Sketch the binding isotherms
log(f/(1-f)) vs log([L]) b) What is the Hill coefficient?
Answer:
a)

b)
nh = 2 / (1+(KD2/KD1))
= 2 / (1+(10-2/10-4))
= 0.18

8)

Two homologs of a protein are isolated. Homolog A is a monomer that binds

glucose with a KD of 4mM. Homolog B is a positively cooperative dimer. The K D of
the first binding site of Homolog B is measured at 4mM. At 1mM concentration of
glucose, it is found that Homolog B binds twice as much glucose as homolog A.
What is the Hill coefficient of Homolog B?
Answer:
For homolog A:
f/(1-f) = [L]/KD = 1/4 = 0.25
Since homolog B, binds twice as much glucose, f/(1-f) = 0.5
f/(1-f) = 0.5 = ([L]/KD1 + ([L]/KD1)* ([L]/KD2))/(1+[L]/KD1)
0.5 = (1/4 + (1/4)* (1/KD2))/(1+1/4) = (0.25 + 0.25* (1/KD2))/(1.25)
KD2 = 0.67mM
nh = 2 / (1+(0.67/4))
=1.41

9)

A cyclist is interested in cheating in a race by delivering more oxygen to his

muscles. The cyclist reasons that since Bisphosphoglycerate (BPG) stabilizes the T
state of hemoglobin, which reduces affinity for oxygen, that reducing the BPG
concentration in his blood cells should be good for his performance. How might
removing BPG might have a detrimental effect on delivery of oxygen to his muscles?
Answer:
BPG acts as an allosteric effector for hemoglobin. Removal of BPG will result in a
sharper switch between saturated and empty hemoglobin. WIthout this effect,
the binding isotherm will not match the difference in oxygen concentration
between the arterial and venous blood and likely lead to suboptimal delivery of
oxygen to his muscles.

10)
Due to a fascinating coupling between folding and ligand binding, the dimeric
acetyl transferase enzyme AAC displays positive cooperativity at low temperatures
and negative cooperativity at high temperatures. At 10C, AAC has a K D1 = 330uM and
KD2 = 130uM. At 37C, AAC has a KD1 = 200uM, KD2 = 230uM. a) What is the Hill
coefficient at each temperature? b) What is the change in fraction bound at 50uM
ligand between 10 and 37C?

(Source: Frieburger et al, NSMB, 2011; Affinities have been altered).

Answer:
a)
For 10C:
nh = 2 / (1+(KD2/KD1))
= 2 / (1+(130/330))
=1.23
For 37C:
nh = 2 / (1+(KD2/KD1))
nh = 2 / (1+(230/200))
= 0.97
b)
For 10C
f/(1-f) = ([L]/KD1 + ([L]/KD1)* ([L]/KD2))/(1+[L]/KD1)
= (50/330 + (50/330)* (50/130))/(1+50/330)
= 0.18
f = 0.18/1.18
= 0.15
For 37C
f/(1-f) = ([L]/KD1 + ([L]/KD1)* ([L]/KD2))/(1+[L]/KD1)
= (50/200 + (50/200)* (50/230))/(1+50/200)
= 0.24
f = 0.24/1.24
= 0.19
Thus, 4% more enzymes are bound at the higher temperature. Even though
AAC is negatively cooperative at higher temperatures, more of the protein is
bound to ligand. This is driven by the favourable change in KD1 at higher
temperatures.
11)
How does CO2 directly and indirectly stabilize the T state of hemoglobin in
venous blood?
Answer:
CO2 directly stabilizes the T state by reacting with the N-terminal amino groups
of the protein. This alters the interfacial interactions between the alpha and beta

subunits, stabilizing the T state relative to the R state. CO 2 indirectly

stabilizes the T state by reducing the pH within red blood cells. The reduction
in pH leads to the Bohr effect where beta subunit His 146 becomes protonated.
This disrupts interactions centred around the ion pair with beta Asp 94 and an
interaction to alpha Lys 40. Without these interactions, the T state is stabilized.

12)
Two proteins are modified by myristoylation, which targets them to the plasma
membrane in a cell at 25C. This changes their effective local concentration from
10nM to 1uM. Assuming that any favourable mutation would increase their
interaction energy by 4 kJ/mol . How many favourable mutations would have to occur
in the absence of co-localization to result in an equivalent effective affinity as
observed when the two proteins are co-localized?
Answer:
G = RT ln(10-8x 10-8)/(10-6x 10-6) = 2.5 * ln (0.001) = -23 kJ/mol
If each mutation added 4 kJ/mol, the at least 6 mutations would be required to
result in as high an effective affinity.
13)
A scientist finds two pathways, dependent on distinct kinase activities, which
activate a stress response in yeast. One pathway responds to elevated levels of salt
and the other responds to elevated levels of caffeine. Both pathways result in
transcription of the chaperone Hsp90. Below is a table listing the relative
concentrations of salt or caffeine and the measured transcription of Hsp90. Explain
which pathway likely contains a single kinase and which pathway likely contains a
kinase cascade similar to the MAP-kinase pathway

[NaCl] mM

Hsp90 transcription

40

[NaCl] mM

Hsp90 transcription

100

10

100

100

100

1000

100

[caffeine] mM

Hsp90 transcription

1.1

10

10

100

50

1000

100
Answer:
Only the salt response is ultrasensitive (in that it undergoes a complete activation
in a ~2-fold rather than ~100-fold concentration change). Ultrasensitivity
resulting from the coupling of multiple kinases converts a graded input signal into
a sharp switch. This situation is observed only for the salt response, which likely
has a kinase cascade, and not for the caffeine response, which is more
hyperbolic.