4 - Protein Structure and Function Myoglobin and Hemoglobin Flashcards
1
Q
Hb and Mb functions
A
Hb transports O2 form lungs to tissue
Mb storage of O2 in sketlal muscle
2
Q
Higher Kd
A
- The more dissociated
- lower affinity (tightness of binding)
3
Q
Lower Kd
A
- more associated / bonded
- higher affinity (tightness of binding)
- more free ligand and protein
4
Q
Which of the following is most likely to explain reversible, concentration-dependent binding of ligands to proteins?
A. H bonds, ion pairs and Van der Waal’s
interactions between the ligand and amino acid side chains
B. H bonds between the ligand and amino acid side chains.
C. Covalent bonds between the ligand and amino acid side chains.
D. The hydrophobic effect
A
A. H bonds, ion pairs and Van der Waal’s
interactions between the ligand and amino acid side chains
5
Q
Higher affinity =
A
Greater % saturation
6
Q
Lower affinity curve will be more towards the
A
Right, with greater Kd
7
Q
Secondary structure of Mb
A
Secondary structure: 8 alpha-helixes (labelled A to H) and some irregular loops.
- no B-sheets
8
Q
Tertiary structure of Mb
A
tertiary structure:
• Includes hydrophobic binding pocket between alpha-helix E and F.
• Includes prosthetic group called Heme
9
Q
Primary structure of Mb
A
Primary structure: 153 amino acids long
10
Q
Structure of heme
A
Porphyrin ring made of 4 Pyrnle sings joined by methylene bridges
- 2 propionyl groups (polar)
- 2 aliphatic/hydrophobic propioynl groups
- Fe2+ held within ring by coordination bonds
11
Q
When heme breaks down, it releases
A
Smaller rings that absorb light
12
Q
Fe2+ ions typically forms
A
6 coordination bonds in an octahedral conformation (two squared-based pyramids, 8 sides)
13
Q
The Fe2+ has 4
A
coordination bonds with N holding it in place in the ring.
14
Q
The 5th and 6th coordination positions are
A
On top and below the Fe2+ whihc form coordination bonds with the coordination position
15
Q
Heme is bound to myoglobin through a
A
coordination bond with the side chain of His F8
16
Q
At the 5th Fe2+ coordination position through
A
Proximal His F8 (8th residue of helix F)
- strong coordination bond formed
17
Q
Heme Forms a Coordination Bond with
A
Proximal His F8
18
Q
Distal his E7allows for
A
O2 to bind to the heme group at the 6th coordination position
19
Q
Distal His E7 causes for O2 to bind to Fe2+ at
A
An angle position because of steric hindress
20
Q
Is the 6th coordination position a true coordination bond?
A
NO, it instead allows for O2 to bind to Fe2+ through a H-bond.
21
Q
Whihc amino acids define the space where O2 will be able to bind to ?
A
Val E11 and Phe CD1
22
Q
The 2 amino acids Val and Phe give prescient of the O2 binding site on Mb, this results in
A
Maximum specificity: determined by shape and space
Maximum affinity: determines by number and type of non covalent interactions
23
Q
Where is the heme prosthetic group held in Mb?
A
In hydrophobic binding pocket
24
Q
O2 is bound to Fe2+ via
A
H-bond
25
Binding site for a ligand on a protein has:
• Precisely defined shape ——>
• Precisely and carefully positioned side groups allow to make specific non covalent interactions. ——>
* Precisely defined shape ——> maximize specificity (which ligands could bind)
* Precisely and carefully positioned side groups allow to make specific non covalent interactions. ——> precise affinity (tightness of binding)
26
Oxygen binds to myoglobin reversibly, with
high affinity
27
Hyperbolic binding curve resents
Fixed High affinity
28
Running on the hyperbolic binding curve causes
%saturation of Mb to decrease and for the reaction to favour the dissociation direction to get into be used in skeletal muscle.
Once its delivered, Mb becomes saturate again and switches favoured side of association
29
Which of the following statements about the oxygen binding site in myoglobin is FALSE?
A. Oxygen binding to myoglobin involves an H-bond with the distal histidine.
B. The heme prosthetic group is held in the heme binding pocket in part via hydrophobic
interactions.
C. The proximal histidine partially blocks the oxygen binding site, forcing oxygen to bind at an angle.
D. The distal histidine helps to define the geometry of the oxygen binding site.
C. The proximal histidine partially blocks the oxygen binding site, forcing oxygen to bind at an angle.
30
Structure of hb
Has quartenary structure
- 2 alpha globins
- 2 beta globins
31
Primary strcure between Mb, a-globin, b-globin is
Very homologous, very little difference.
32
Types of amino acid substitution
1) conservative substitution: staying the same
- structural / properties the same
20 critical substitution: changing dramatically, effect protein structure and function
33
Strcure of all the globin in terms of primary, secondary and tertiary
They are all pretty much identical
34
Both alpha and beta globin have
- 8 a-helixes
- similar irregular loops
- heme binding pocket with hydrophobic bottom
- both have proximal His F8 and distal E7
- F8 binds to Fe2+ and E7 defines O2 BS at angle
35
When Mb binds to O2 = Hb does what
When Mb releases O2 = Hb
When Mb binds to O2 = Hb releases O2
When Mb releases O2 = Hb binds to O2
36
Mb and Hb must have different
Kd values for them to function
37
Sigmoidal binding curve
Represents change in binding affinity
- shows protein affinity changing
- higher Kb
38
Myoglobin and hemoglobin have different oxygen binding behaviour.
How does Hb (or any protein) alter its affinity for its ligand?
By changing the shape of the binding site and non covalent interactions
39
Affinity / specificity of a ligand binding site is determined by:
1. The shape of the binding site, which is determined by
- distal His E7 ( causing O2 to bind at angle)
- two non polar hydrophobic side chains (Val and Phe) that point into the binding site and restrict the space available.
2. Non covalent interactions of the binding site.
• H-bond between O2 and distal E7
40
• Because tertiary folded structure is stabilized by only non covalent interactions. There is the ability to
move atoms slightly, break the non covalent interaction, move atom slightly, then form another non covalent bond to change the precise shape of the binding site for the ligand, resulting in a change in affinity.
41
How to hb chnage its affinity for O2
Small changes in the tertiary structure whihc changes the shape of the ligand binding site ‘ changing the Hb affinity and specificity of binding
42
2 conformations of hb
OxyHb and deoxyHb
43
DeoxyHb
- larger central cavity
- interface between alpha/beta subunit 44 and 41
- Thr,Pro
- tensed
- lower affinity
44
OxyHb
- relaxed state
- interface between 38 and 41
- thr,thr
- higher affinity
- smaller cavity
45
Chnage from deoxyHb to Oxyhb is a chnage in
Quartenary structure
46
A chnage in quartenary strcure between deoxyHb and oxyHb is driven by
Tertiary structure of each subunit
47
Hemoglobin is an
Allosteric protein
48
Ligands that cause conformational change in allosteric proteins are called
Allosteric effectors
49
Allosteric effectors
Are a shape and cause other shapes
50
• Allosteric activators favour the
relaxed state. Favour the reaction of going firm tensed to relaxed state.
(Positive allosteric effector)
51
• allosteric inhibitors favours the
tensed state. Favour the reaction going from relaxed to tensed.
(Negative allosteric effector)
52
• homoallosteric effectors:
they change the proteins ability to bind themselves, they alter their own binding affinity.
53
• Heteroallosteric effectors:
they alter the binding affinity for something else or another ligand. (Protein that has many ligands)
54
O2 is a
homoallosteric activator
55
H+ and BPG are both
Heteroallosteric inhibtors
56
Oxygen is the substrate for
Hb, Hb binds transports oxygen to transports it. So oxygen’s Hb’s substrate. It effects the binding of oxygen.
57
When hb goes back inot the lungs its in its
Tensed state
58
When hb goes inot the tissues you goes into
Relaxed state and when it leaves it returns back to tensed state by the time it gets to the lungs again
59
• If you increase oxygen concetration, reaction will shift to favour the
production of oxyHb, favours forward reaction/bound O2.
60
The O2 being an allosteric activator for Hb mechanism works because of the conformational changes that occur to the
3 tertiary structure of the subunit when O2 binds.
61
How do conformational changes to the oxygen binding to hemoglobin occur.
Fe2+ pops out of the plane of the ring when theres nothing in the O2 binding site.
- when O2 binds to the BS, the H-bond pulls the Fe2+ back into the plane of the ring, equalizing with the proximal His F8 coordination bond on the other side.
62
Conformational chnage mechanism
* O2 pulls the Fe2+.
* Fe2+ pulls the proximal His F8
* Proximal His F8 pulls alpha helix F.
63
Why does deoxyHb shatter when O2 binds to it?
Because of the conformational chnage that occurs when O2 pulls the Fe2+ ION INOT THE PLANE, whihc pulls the F8 which pulls the helix = changing the tertiary structure of the subunit so much that the whole crystals will shatter.
64
Hb is more likely than Mb to release O2 in resting muscle?
Yes
65
BPG and H+ ions favour formation of the
Tensed state of hemoglobin
66
Equilibrium postion (the ratio of T and R state), depends on the concentration of
H+ ions and the presence of BPG. The more H+ ions you have, the more your gonna be in the tensed state of the protein.
67
• without BPG. The tensed state
cannot exist. BPG is absolutely essential, therefore essential for oxygen delivering in the blood.
68
2,3-Bisphosphoglycerate (BPG) is essential in stabilization of the T state of Hb.
What are its properties?
* Produced from an intermediate of glycolysis.
* Very important molecule
* Small organic molecule.
* Very polar
* Highly negatively charged.
* Found in really high concentrations in red blood cells, where the Hb NEEDS it to adopt the tensed state.
69
BPG binds in the
central cavity of deoxyhemoglobin
70
How does BPG bind to the central cavity of deoxyHb?
Through salt bridges, electrostatic interactions
71
With what does BPG form salt bridges with?
• forms salt bridges with amino acid side chains that are positioned into the cavity (pointing into the cavity).
72
• the side chains are ______ charged because BPG is ______ charged.
positively
negatively
73
The interaction of BPG occurs in the presence of whihc amino acids?
Histidine ((+) or neutral) and lysine (always +)
74
• N-terminals of polypeptide is always
positively charged. Point into the central cavity. And are involved in electrostatic interactions / salt bridges with PBG.
75
• when PBG forms all those salt bridges, it
locks the protein in its tensed state (or helps stabilize it in its tensed state, as tensed state is uncomfortable and would rather be in relaxed state). So needs to be pushed into tensed state, does this happens through the binding of PBG.
76
H+ ions facilitate formation of the
Tensed state
77
• H+ ions at produced by
metabolically active tissues (which acts as a single to favour the formation of the tensed state and therefore increase oxygen delivery)
78
• CO2 Is also produced when we are metabolically active in the
citric acid cycle of glycolysis, CO2 is solubilized in our blood stream and tissues to form carbonate and H+ ions.
79
• When H+ ions are produced metabolically, they are produced during the hydrolysis of ATP as
muscle contract. The driving force to make muscle contract is the hydrolysis of ATP. As muscle consume lots of ATP to contract, they release a lot of H+ ions.
80
• an increase in H+ ions =
decrease in pH. (Inverse relationship)
81
• When His+ is in the central cavity, affinity for BPG increases. The KEY FACTOR of the mechanism for how
H+ ions work. They make sure the His are all positively charged, whihc increases BPG binding affinity to the Hb.
82
When H+ ions are high its causes the pH to decreases which results in BPG
Affinity increasing = favouring the tensed state = curve moves to the right with larger Kb and lower saturation.
83
When H+ ions are low = pH is high (basic) and
Greater affinity for oxygen binding to Hb = Meaning you have less BPG = The reaction will shift to favour the relaxed state.
84
• when you generate the H+ ions the percentage saturation at any concentration of oxygen
decreases. So your delivering oxygen. Important for muscle.
85
• The release or production of H+ ions enhances
oxygen delivery, cuz it reduces the affinity of Hb for O2 And allows more oxygen to dissociate.
• Works good as a signal mechanism to provide muscle with the oxygen they need.
86
Which of the following is true regarding the ability of Hb to bind oxygen?
A. The presence of CO2 promotes the release of oxygen.
B. Salt bridges stabilize the deoxy form of Hb. C. H+ and BPG stabilize the deoxy form of Hb.
D. B and C are true.
E. All the above are true.
E. All the above are true.
87
• Under any given time, a proportion will be in tensed state, and some will be in relaxed state. this depends on the combined effects of
oxygen concentration, BPG, and pH.
88
BPG has
Variant affinity
89
What’s the affinity of BPG dependant on?
H+ ions concentration
90
When H+ ions are high BPH binds at
High affinity
91
• BPG is absolutely required to
stabilize the tensed state by binding to the central cavity. When Hb switched form tensed to relaxed the BPG is squished out as the protein.
92
• Critical amino acids:
proximal His and the distal His are both critical in oxygen binding, essentially determine the function of the protein cuz without them they cannot function.
93
Single amino acid changes:
1. Physiological / deliberate changes : fetal Hb
2. Mutation: causes a disease —-> disciple cell anemia
• both occur because of a single amino acid change in the protein structure.
94
Fetal Hb
Has gamma globin instead of B-globin
95
What amino acid chnage occurs for fetal Hb to exist
His 143 —->Ser 143
Large change as His is (+) charged and Ser is uncharged
96
The replacement of His with Ser causes the affinity of BPG to
Decrease
97
How does der effect BPG
• This causes fewer salt bridges to form and unstable BPG and therfore unstable tense state of the Hb.
98
• The fetus gets its oxygen from maternal circulation in placenta. Fetal blood takes oxygen from maternal Hb. Therfore fetal Hb requires
higher affinity for oxygen so it can steal oxygen from maternal Hb. All cuz relaxed state is favoured.
99
• Sudden fetal death syndrome:
babies are supposed to stop making gamma globin when they are born, and switch to adult beta Hb. if they fail to do so, they have way to high of an affinity for oxygen in relaxed state whihc prevents proper O2 delivery in tissues. Tensed state isn’t stabilized enough. Improper O2 delivery.
100
• Sickle cell due to single amino acid mutation in the
beta-globin gene where residue number 6, which is a Glu6 is mutated to Val6.
101
The amino acid chnage of glu6 to val6 causes what
Formation of hydrophobic patches on surface or B-globin
102
How do these hydrophobic patches cause aggregation and fiberilization of the RBS to distorted their shape?
Hydrophobic Val put itself into the hydrophobic pocket causing fiberilization and distorts the shape of the RBC
103
What drives the aggregation of sickle cell RBC
Hydrophobic effect drives Hb fibres to form
104
Main roles of His in Hb
1. Forms salt bridges to bind BPG to bind to Hb
2. Used in Hb to stabilize tensed state
3. Distal histidine E7 used to angle the bond of O and Fe2+
4. Proximal histidine F8 used to form coordination bond with 6th coordination position
105
```
The R state is the favoured form of Hb in the lungs
A. True
B. False
C. The R and T state are present in
the same ratio in the lungs
```
B. False
106
Did you have fun
Of cuurse you didn’t go fuck yourself :)
