hemoglobin: a paradigm for cooperativity and allosteric regulation
TRANSCRIPT
Hemoglobin:A Paradigm for Cooperativity and
Allosteric Regulation
Why do we breathe?
http://www.uni.edu/schneidj/webquests/spring04/tvbroadcast/circulatorysystem.html
Cellular Requirement for O2
Catabolism
(Oxidation)
O2
ADP
ATP
NADP+
NADPH
Intermediates
Anabolism
(Biosynthesis)
ProteinsFats
Carbohydrates(Nutrients)
Waste
(CO2/ Urea/ etc.)
Oxygen Carriers
Diffusion
Limited solubility of O2 in Blood and Cell Water
Myoglobin and Hemoglobin
• Myoglobin (Mb)– Increases O2 solubility in tissues
(muscle)
– Facilitates O2 diffusion
– Stores O2 in tissues
• Hemoglobin (Hb)– Transports O2 from lungs to peripheral
tissues (erythrocytes)
Oxygen Transport
O2
O2 O2
deoxyHb
deoxyMbMbO2
Hb(O2)n Hb(O2)n
deoxyHb
deoxyHb
LUNGS MUSCLE CELL
pO2 = ~20- 30 torr
RED BLOOD CELLS
O2 + 4e– + 4H+ 2H2O
pO2 = 100 torr
Myoglobin
Small Intracellular Protein in Vertebrate Muscle
Function(s) of Myoglobin
Facilitate O2 Diffusion in Muscle
O2 Storage (aquatic mammals)
Figure 7-1
Structure of Sperm Whale Myoglobin
Figure 7-2
The Heme Prosthetic Group
Properties of Heme Prosthetic Group in Myoglobin
• Tightly bound
• Synthesized separately from myoglobin
• Fe2+ Coordination– Nitrogens of heme (4)
– His (F8): proximal histidine
• His (E7): distal histidine
• Ligands: O2, CO, and NO
Ligands
Small molecules that bind to proteins by non-covalent
interactions(e.g. O2 to myoglobin)
Ligand Binding
•usually transient and reversible interaction with others molecule (= ligands) such as metals, hormones
•often involves “molecular breathing” of the protein, i.e. ability to undergo small conformational changes
•often induces molecular rearrangements in the protein
• ligand binding sites are- highly conserved- complementary in size, shape, and charge
• prosthetic (permanent, non-proteinaceous)
•group of Mb and Hb
• incorporated into Hb and Mb during folding
• responsible for reversible O2 binding
• responsible for red color of blood and muscles
Heme
central Fe2+
Heme – Structure
2 vinyl groups (buriedin protein)
4 methyl groups
2 propionate groups(exposed)
•Fe2+ has 6 coordination sites
•4 with N of pyrrole rings,
•2 perpendicular to ring
•Mb/Hb: 5th coordination site is occupied with proximal His
•6th coordination site:O2 oxyhemoglobinnone deoxyhemoglobinCO carboxyhemoglobin
Heme – Iron Coordination
Heme – Binding of CO vs. O2
• free heme binds C0 105 times better than O2
•kinked binding topology in Mb/Hbfavors O2 (100-fold)
TOTAL: CO binding ~ 230 fold stronger than O2 binding (Carbon monoxide poisoning)
Function(s) of Myoglobin
Facilitate O2 Diffusion in Muscle
O2 Storage (aquatic mammals)
Myoglobin (Mb)
•primarily found in muscle (highly abundant in marine mammals such as whales)
•single polypeptide (153 aa) with one bound heme
•very simple oxygen binder: binds oxygen at high pO2, releases it at low pO2
Mb + O2 MbO2
• typical globin fold
8 helices (A-H) and loops in between
20MCDB310 – Chapter 5: Protein Function
The Globin Fold
Myoglobin – Oxygen Binding Curve
Binding/Association Constant Ka
Quantitatively describes the affinity of a protein P for its ligand L
P + L PL
the higher the binding affinity, the higher Ka
[L][P]
]PL[
aK
Dissociation Constant Kd
P + L PL
the higher the binding affinity, the smaller Kd
]PL[
[L][P]1
ad KK
Example: Ka = 106 M-1 Kd = 10-6 M
Degree of Saturation,
0 1
[P][PL]
[PL]
]sites binding total[
sites] binding [occupied
Fraction of binding sites that are occupied by ligand at any given ligand concentration
Degree of Saturation,
Using
[L]
[L]
[L]1
[L]
da
a
KK
K
If [L] = Kd = 0.5
Kd is the ligand concentration at which 50% of the binding sites are occupied
[L][P]
]PL[
aK [L][P][PL] aK
Ligand Binding Curve
[L]
[L]
d K
Some Examples
with KD = 1 µM
Question: What fraction of the protein has ligand bound when the [L] is 1 µM or 10 µM?
[L] = 1 µM:
[L]
[L]
d K
5.0μM1μM1
μM1
[L]
[L]
d
K
[L] = 10 µM: 91.0μM10μM1
μM10
[L]
[L]
d
K
Some Examples for Dissociation Constants
Myoglobin – Oxygen Binding Curve Revisited
[L]
[L]
d K
When ligand is a gas, partial pressures = concentrations
250
2
O
O
pp
p
Saturation of Mb depends on
•the binding constant of Mb for O2 (KD = p50 = 2.8 torr)
•the concentration of O2 (pO2)
Question: What is the fractional saturation of Mb?
pO2 = 1 torr:
pO2 = 10 torr:
Myoglobin – Oxygen Binding Curve Revisited
26.0torr8.2torr1
torr1
78.0torr8.2torr10
torr10
[L]
[L]
d K
pO2 in tissue ~ 4 kPa
Myoglobin – An Oxygen Storage!
pO2 in lung ~ 13 kPa
10 kPa = 76 torr
Hemoglobin(22)
Hemoglobin (Hb)
•present in erythrocytes (makes blood look red, 34% of weight is Hb)
Different Hb subtypes:•Hb A (adult): two (141 aa) and two (146 aa)
subunits that are arranged as a pair of identical subunits (2 subunits)
•Hb F (fetal): two and two chains
12
2 1
Hemoglobin – 3D Structure
Each subunit has 1 heme, which binds 1 O2
Lehninger, Figure 7-5, 7-6
O2
Heme
Hemoglobin
Erythrocytes:
•1 ml blood: 5 x 109 erythrocytes•1 erythrocyte: 3 x 108 Hb molecules •Hb is a good marker for number of red blood cells
Homology:
• 50% of AA are identical between and subunits
• 20% of AA are identical between / and Mb
Function of Hemoglobin
O2 binding in lungs
O2 release in tissues
Oxygen Transport
O2
O2 O2
deoxyHb
deoxyMbMbO2
Hb(O2)n Hb(O2)n
deoxyHb
deoxyHb
LUNGS MUSCLE CELL
pO2 = ~20- 30 torr
RED BLOOD CELLS
O2 + 4e– + 4H+ 2H2O
pO2 = 100 torr
Oxygen binds to hemoglobin and myoglobin differently
Myoglobin
Hemoglobin
Oxygen binding to hemoglobin
Θ = fraction of binding sites that are occupiedpO2 = partial pressure of oxygen
p50 is the pO2 where half the binding sites are occupied
p50
Hb has evolved to transport O2
pO2 In Lungs
pO2 In Tissues
p50
38%
Hb gains cooperativity by switching between 2 states
Lehninger Figure 7-10
T state (Low Affinity) R state (high affinity)
The Concerted ModelAll or nothing mechanism
T RLehninger, Figure 7-14
The Concerted ModelAll or nothing mechanism
T RLehninger, Figure 7-14
The Sequential Model
Hb follows a little of both
T RLehninger, Figure 7-14
Figure 7-8
Movements of the Heme and the F Helix During the T —> R Transition
Local structural changes around the Heme are communicated to the rest of Hb
By Janet Iwasa,https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html
Figure 7-9
Changes in the 1–2 Interface during the T —> R Transition in
Hemoglobin
Figure 7-9 part 1
Changes in the 1–2 Interface during the T —> R Transition in
Hemoglobin
Figure 7-9 part 2
Changes in the 1–2 Interface during the T —> R Transition in
Hemoglobin
Figure 7-10
Networks of Ion Pairs and Hydrogen Bonds in Deoxyhemoglobin
T vs R State
(1) Change at interface between and
(2) R state is more compact, and relaxed(3) T state has additional salt bridges, which makes it more tense
(4) In R state individual O2 sites have higher affinity for O2.
- better Fe-O2 bond length - fewer steric repulsions associated
with oxygen binding.
Without cooperativity Hb could not efficiently transport oxygen
T state
LungsTissues
homotropic, positive (= cooperative binding)
Allosteric regulation of protein function
homotropic, positive (= cooperative binding)
Allosteric regulation of protein function
heterotropic, negative
The Bohr Effect
• H+ and CO2 are negative, heterotropic modulators of Hb
• metabolizing tissue: H+ and CO2 accumulate bind to Hb and lower the affinity of Hb for O2
Hb releases O2
• lungs: CO2 and H+ dissociate from Hb increases the affinity of Hb for O2
Hb binds O2
• increase the efficiency of Hb as O2 transporter
Hb also binds and transports H+ and CO2 from tissue to lungs and kidneys for secretion
The Bohr Effect
Lungs: pO2 = 100 torr, high pH (7.6), low [CO2] Hb has high affinity for O2
Tissue: pO2 = 20 torr, low pH (7.2), high [CO2] Hb has low affinity for O2
CO2 + H2O HCO3- + H+
CO2 + H2O HCO3- + H+
Bohr effect
pH Dependence of O2 Binding to Hb
Mechanism of Bohr Effect1. Protonation of His-146
His-146+ forms salt bridge with nearby Asp-94 stabilizes low affinity T-state
O2 is released as pH drops
61MCDB310 – Chapter 5: Protein Function
Figure 7-12
Roles of Hemoglobin and Myoglobin in O2 and CO2 Transport
Heterotropic Negative Modulator
Or: 2,3-Diphosphoglycerate (DPG)
BPG is negatively charged
BPG binds to the central cavity of Hb
BPG binds to the positively charged central cavity of Hb
By Janet Iwasa,https://iwasa.hms.harvard.edu/project_pages/hemoglobin/hemoglobin.html
BPG allows for release of O2
pO2 In LungsAt Sea Level
pO2 In Tissues
No BPG
5mM BPG
In Class Activity:
Ligand Binding can affect Protein Function
• Cooperativity– 1 ligand bound = higher affinity for more ligands– Concerted vs Sequential
• Allosteric regulation– 1 regulator binding affects binding of ligand – Homotropic vs heterotropic– Positive vs Negative
From Protein Structure to Function
1. Hemoglobin and myoglobin: Principles of reversible ligand binding
2. (Antibodies: Principles of specific, high affinity ligand binding)
3. Myosin and actin: Protein activity modulated by ATP
4. Enzymes
Table 7-1
Hemoglobin Variants
Sickle Cell anemia
• Glu ——> Val (residue 6 of -chain)
• Leads to hydrophobic interactions between hemoglobin molecules
• Hemoglobin fibers
• Sickling of erythrocytes
• Increased resistance to malaria
Figure 7-17a
Normal Erythrocytes
Figure 7-17b
Sickled Erythrocytes
Figure 7-20
Correspondence between Malaria and Sickle-Cell Gene