Lecture 8C Flashcards
1
Q
What are five possible molecular structures of DNA? Quick characteristic of each
A
- Primary (sequence)
- Secondary (local folding)
- Tertiary (long-range folding)
- Quaternary (multimeric organization)
- Supramolecular (large-scale assemblies)
2
Q
Why are myoglobin and hemoglobin critical proteins for our survival?
A
O2 is not very soluble in aqueous solutions like blood and can’t be transported freely to tissues – it also does not diffuse well across tissues – must be transported to the tissues, stored there until needed.
Myoglobin and hemoglobin are critical for oxygen transport and storage
3
Q
What are good examples of various levels of protein structure?
A
Myoglobin and hemoglobin
4
Q
What good example of various regulatory strategies, such as cooperativity (in oxygen binding) and
allosteric contro?
A
Myoglobin and hemoglobin
5
Q
Role of globins in oxygen transport & storage?
A
Myoglobin and hemoglobin provide tissues with a continuous oxygen (O2) supply
6
Q
Hemoglobin is used for ____ in all vertebrates and some invertebrates?
A
Oxygen transport
7
Q
myoglobin is the _____ used in all animal species
A
oxygen storage protein
8
Q
hemoglobin also ____ from tissues
A
Removes CO2
9
Q
What is the “ligand” for hemoglobin and myoglobin?
A
O2
10
Q
What is PO2?
A
partial pressure of O2 (a measure of dissolved O2 concentration).
11
Q
For iron-binding proteins, P50 represents what?
A
the partial pressure at which 50% of the ironbinding sites (hemes) have O2 bound
12
Q
Where is there high PO2?
A
Lungs
13
Q
Where is O2 released
A
• release in capillaries at tissues (moderate to low PO2) or store until required
14
Q
During pregnancy how does the fetus get O2?
A
during pregnancy the fetus must get O2 from the mother’s bloodstream - uses a
special (fetal) hemoglobin
15
Q
Compare and contrast myoglobin and hemoglobin
A
Myoglobin – oxygen storage • present in tissues (muscles) • monomer • high affinity for O2 • unaffected by pH, [CO2] or [BPG]* • binds 1 O2 molecule • doesn’t bind 2,3-bisphosphoglycerate* (BPG)
Hemoglobin – oxygen transport • present in blood • tetramer: 2 alpha, 2 beta subunits • moderate affinity for O2 • sensitive to pH, [CO2] and [BPG] • binds 4 O2 molecules
16
Q
Where are myoglobin found?
A
globular protein founds in muscles
17
Q
How many AA in myoglobin
A
153
18
Q
% made up of alpha helices? How many types of a-helices
A
77%
eight a-helices: A, B, C, D, E, F, G, H
19
Q
Are the exterior and interior of myoglobin AA’s polar or non polar?
A
- interior residues are non-polar except residue 7 of helix E (His E7) and His F8, which bind the heme group
- exterior residues include both polar and nonpolar
amino acids
20
Q
What does a myoglobin’s heme prosthetic group consist of?
A
- consists of porphyrin and Fe2+ ion
21
Q
What is the heme group required for
A
- tertiary structure and O2 binding
- heme binds O2 via the Fe2+ ion
22
Q
What is an apoprotein aka apomyoglobin
A
myoglobin without heme
23
Q
What is an apomyoglobin + heme
A
Myoglobin
24
Q
What is an protoporphyrin IX
A
tetrapyrrole ring system
25
protoporphyrin IX + Fe2+ = ?
Heme
26
Iron-storing transport molecules must be able to do what 3 things
must be able to bind O2, not allow it to oxidize to any other substance, and release it on demand
27
What is responsible for the distinct red color of blood and muscles?
Heme
28
The capacity of globins to bind oxygen depends on the presence of what?
Bound heme
29
Where is the heme prosthetic group located?
Wedged between the hydrophobic E and F a-helices
30
There are 2 histidines involved in coordinating the heme iron for both myoglobin and hemoglobin. How do their roles differ?
His F8 coordinates the heme Fe2+ , while HisE7 stabilizes O2 with a hydrogen bond when the oxygen provides a 6th ligand and is bound.
31
What keeps the iron in the reduced form?
The hydrophobic environment of the protein in the heme binding site keeps the iron in a reduced (Fe2+) form
32
What is P50?
the PO2 at which half the myoglobin molecules have O2 bound (Y = 0.5).
33
What is the P50 for human myoglobin? Is it high?
For human myoglobin, P50 = 2.8 torr: Mb binds O2 with high affinity.
34
What happens at 20-30 torr?
At 20-30 torr, as in tissues, virtually all the myoglobin molecules have bound O2. i.e., myoglobin is saturated.
35
What is the pO2 in the lungs?
~100 torr
36
What can carry more oxygen than water?
Blood
37
How many subunits does hemoglobin have? What are they? How do they compare to myoglobin
4 subunits
- 2 a subunits (141 amino acids each)
- 2 b subunits (146 amino acids each)
- the are structurally homologous to myoglobin
38
How can hemoglobin be considered as a dimer of ab dimers?
(denature Hb in urea -> ab dimers
39
Although each a and b subunit individually looks like myoglobin, why are they actually very different?
only 27 amino acids are conserved (identical) among myoglobin and a and b subunits of Hb
40
What causes the differences in function between Hb and Mb
all due to the quaternary structure of Hb... causes:
• cooperative binding to O2
• allosteric regulation by CO2, H+, and BPG (DPG)
41
Do Mb and Hb alpha and beta subunits bind to their heme prosthetic group the same way?
Yes
42
Which ones are more similar to each other, Hb alpha and beta subunits or Hb alpha & myoglobin or Hb beta* myoglobin?
Hemoglobin a and b subunits are more similar to each other than to myoglobin
43
How does hemoglobin bind to oxygen?
Loosely and reversibly
44
What colour is Hb bound to O2?
Bright red
45
Why does Hb transports oxygen efficiently?
By binding oxygen cooperatively
46
What does oxygen binding of one or 2 hb subunits alter?
The quaternary structure of hb, increasing the affinity of the other subunits for oxygen (aka positive cooperatively)
47
How does the state of the complex change after binding of oxygen?
Binding of oxygen to one or two hemoglobin subunits (chains) results in a conformational change of the entire complex from a “tensed state” (T state) to a “relaxed state” that dramatically enhances oxygen binding to the remaining subunits - allosteric control between the subunits of hemoglobin.
48
Structural changes in hemoglobin are brought on by?
O2 binding
49
Prior to binding O2, how is the Fe2+ positioned as well as the heme.
Prior to binding O2 the Fe2+ is outside the plane of the heme and the heme is slightly “puckered”.
50
After O2 binds, how is the Fe2+ positioned as well as the heme
Upon oxygenation the iron ion moves into the plane of the heme group, pulling with it the coordinated His F8, which in turn pulls the F helix toward the heme
51
Where does the carboxyl terminal end of the F helix lie?
The carboxyl terminal end of the F helix lies at the interface between the two ab dimers. Consequently, the structural transition at the iron ion is directly transmitted to the other subunits.
52
Is the T or R state the deoxy or oxy?
T is deoxy, R is oxy
53
Does the T or R state have increased affinity for O2?
R state
54
the R state differs from the T state by a rotation of about?
the R state differs from the T state by a rotation of about 15 degrees of the alpha1beta1 dimer (dark colours) with respect to alpha2beta2 together with a shift that brings the b subunits (blue) closer together and narrows central cavity
55
Why is the cooperative binding in hemoglobin very efficient?
it permits full saturation of the protein in the lungs (or gills), where PO2 is high and efficient O2 release in the tissues, where PO2 is low
56
Reason why the cooperative binding in hemoglobin is very efficient?
due to the existence of a cooperative or allosteric interaction among the O 2 -binding sites in the hemoglobin molecule, i.e. the filling of the first site (or more likely first two sites) increases the affinity of the other sites for O 2 - initial binding acts like a switch.
57
What are the two states of hemoglobin
deoxyhemoglobin (zero O 2 bound) and oxyhemoglobin (4 O 2 bound) - is either fully loaded or unloaded - not much of an intermediate.
58
Why is cooperative binding not observed in myoglobin?
Myoglobin is a single subunit. Cooperative binding requires “communication ” between the different hemoglobin subunits regarding their oxygenation state (filled or empty); it is only possible because of the quaternary structure of the protein.
59
PO2 conditions that allow hb to bind?
• hemoglobin must be able to bind oxygen under conditions of high PO2 (in the lungs) and release it under low PO2 (in the tissues)
60
What curve shape indicates cooperatively?
sigmoidal shape
61
if O2-binding was not cooperative, what possible PO2 levels for bind and release be?
if O2-binding was not cooperative, O2 would either bind well at high PO2 but not release well at low PO2, or release well at low PO2 but not bind well at high PO2
62
If O2 binding was cooperative, what are the levels of PO2 for bind and release
• cooperativity means that hemoglobin has a high affinity for O2 at high PO2 in the lungs and a low affinity for O2 in the low PO2 of the tissues (it has released 66% of its O2 at 20% PO2) – notice the sigmoidal shape of the curve, indicating cooperativity
63
At what levels does myoglobin bind and release?
Myoglobin, on the other hand, must bind to O2 at PO2 where hemoglobin releases it, and release it at really low PO2.
64
The difference in O2-binding affinity between hemoglobin and myoglobin ensures?
that O2 bound to hemoglobin in the lungs is released to myoglobin in the muscles (tissues).
This oxygen delivery system is efficient because the tissue PO2 corresponds to the part of the hemoglobin binding curve where the O2 affinity falls off the most sharply
65
Heme binds O2 weakly and reverisbly but what compounds strongly?
gases like carbon monoxide (CO), hydrogen sulfide (H2S) and ions like cyanide (CN- ) very strongly.
CO, H2S, CN- are very toxic – they bind to hemoglobin and block O2 binding.
66
Other factors affecting the O2 affinity of hemoglobin – conditions in tissues facilitate unloading of O2
• BPG (2,3-bisphosphoglycerate): lowers O2 affinity of hemoglobin (but not
myoglobin)
• reduction in pH (found in respiring cells) causes lower O2 affinity of hemoglobin
• release (accumulation) of CO2 in respiring tissues lowers O2 affinity of hemoglobin
67
What factors help stabilize the deoxygenated T state of Hb? This in turn promotes what?
Higher concentrations of H+ and CO2 in respiring tissue help to stabilize the deoxygenated T state of Hb, which promotes the release of O2 and works against rebinding of O2.
68
What is BPG?
2,3– bisphosphoglycerate (BPG), an isomer of an intermediate in glycolysis
69
Where is BPG found? What concs?
RBCs, same conc as hemoglobin
70
How does BPG affect oxygen affinity so significantly?
BPG is a highly negatively charged compound that binds to positively charged side chains and N-terminal amino groups in the center of the hemoglobin tetramer. BPG has a higher affinity for the deoxygenated T form of hemoglobin. As the RBC gets into the vicinity of tissues (esp. respiring cells) it offloads O2 and is rapidly bound by BPG. BPG-binding stabilizes the T form, preventing O2 from re-binding. The presence of BPG makes hemoglobin more efficient at unloading O2 in the tissues.
71
A single molecule of BPG binds to ?
central pocket in the hemoglobin tetramer
72
What form of hemoglobin does BPG prefer to bind to?
BPG binds
preferentially to deoxyhemoglobin (T form), because in the oxy form (R form) the pocket is
too small to accommodate this molecule.
73
What does the binding of BPG cause?
Bound BPG stabilizes the deoxy form, reducing the oxygen affinity of hemoglobin, which promotes the release of any bound O2 and prevents further binding of O2. Thus, the presence of BPG makes hemoglobin more efficient at unloading O2 in tissues. As a result, dissociation of O2 from hemoglobin in tissues is enhanced
74
What are the levels of BPG in pregnancy? Causes?
BPG levels are elevated during pregnancy. BPG allows fetal hemoglobin to compete with maternal hemoglobin for O2 because fetal hemoglobin has a lower affinity for BPG.
75
How does the hemoglobin tetramer in fetals differ from adults?
fetal hemoglobin tetramers include two a chains and two g chains. g chains are 72% identical in amino acid sequence with the b chain, but His143, which is critical in binding BPG, is substituted with a serine.
76
What does the substitution of His143 to serine in fetals cause?
This change removes positive charges from the BPG binding site and reduces the affinity of BPG for fetal hemoglobin, thereby increasing the oxygen-binding affinity. This allows oxygen to be effectively transferred from maternal to fetal RBCs, because the fetal hemoglobin could pick up O2 released by maternal hemoglobin.
77
P50 for fetal homoglobin is higher or lower than maternal hemoglobin?
(50% saturation occurs at a lower PO2 for fetal hemoglobin than for maternal hemoglobin.)
78
The T state (deoxy) of Hb is stabilized by 2 salt bridges between which 3 amino acid residues
a2 Lys40, b1 His146 and b1 Asp94
79
One of the two salt bridges depends on b1 His146 being
Protonated
80
In actively metabolizing cells, CO2 is produced and converted to bicarbonate, ____ the pH in tissue
decreasing
CO2 + H2O ->
81
What does the low pH allow?
The low pH helps to protonate b1 His146, stabilizing the T state and favoring release of any remaining bound O2
Protonation of Hb helps to transport excess H+ to the lungs and kidneys.
82
Conversely, increasing the pH (removing protons) does what?
stimulates Hb to bind more O2 at low PO2.
83
In tissues, much of the H+ generated by the conversion of CO2 to bicarbonate is taken up by hemoglobin in the ___ state? This drives the equation to the right or left
- T
| - This drives the CO2 conversion reaction to the right.
84
Most CO2 produced in the tissues is carried to the lungs in the form of ___ - . However, some CO2 can bind to _____ of each hemoglobin subunit to form ____
Most CO2 produced in the tissues is carried to the lungs in the form of "HCO3 -" . However, some CO2 can bind to the "a-amino group at the N-terminus" of each hemoglobin subunit to form "carbaminohemoglobin"
85
What does the formation of carbaminohemoglobin: result in? Effects?
produces H+, which can bind to hemoglobin and stabilize the T state. In addition, the negatively-charged carbamate can form salt bridges with positively charged side chains on hemoglobin, further stabilizing the T state and promoting the release of O2.
86
Hemoglobin can transport CO2 in the form of _____ to the lungs where the ____ O2 concentration and ____ [CO2] and [H+] promotes O2 ____ and ___ of CO2.
Hemoglobin can transport CO2 in the form of "carbamate" to the lungs where the "high" O2 concentration and "low" [CO2] and [H+] promotes O2 "binding" and "release" of CO2.
87
What is the Bohr effect?
The decrease in O2 affinity of hemoglobin in the tissues due to high concentrations of H+ and CO2, and the increase in O2 affinity in the the lungs due to low concentrations of H+ and CO2
88
What is a disease connected with hemoglobin
Sicke-cell anemia
89
What causes sickle cell anemia?
- results from an autosomal recessive mutation in the gene encoding the hemoglobin b chain, resulting in a single amino acid change: Glu6 -> Val
- negative charge is changed to a nonpolar (hydrophobic) group
- RBCs lyse leading to anemia (loss of RBCs in blood, reduced ability to carry O2)
90
Where is the mutation?
• this mutation lies at the surface of the protein in the deoxy (T) form of hemoglobin -> deoxyhemoglobin S
91
What causes the sickle-shaped cells?
this results in the b chains in the deoxy form ‘sticking’ together and forming fibers that cause the red blood cells to elongate -> sickle-shaped cells
92
How do normal red blood cells and sickle cells differ?
In sickle-cell disease, RBCs become sickle-shaped w/reduced elasticity. They can't pass thru capillaries easily and occlude them, blocking blood flow. capillary rich organs experience impaired
circulation which can result in organ damage
93
___ [O2] causes a sickling crisis
Low [O2]
