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Chem 474 Biochemistry l > Chapter 3 Binding and Molecular Recognition > Flashcards

Chapter 3 Binding and Molecular Recognition Flashcards

1
Q

What do both myoglobin and hemoglobin bind to?

A

Heme

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2
Q

Function of myoglobin vs hemoglobin

A
  • myoglobin: binds oxygen from blood + stores it for when muscles expend energy or need to convert molecules from food into useable forms
  • hemoglobin: found in blood, transports oxygen from lungs to tissue
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3
Q

What is L1/2?

A

The concentration of L (ligand) at which half the receptors are bound to L and half are free

when half of the receptors (R) are bound to a ligand (L)

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4
Q

Molecular recognition

A

The ability for certain molecules to bind to one another
- forms complexes w/ particular 3D shapes and well defined properties

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5
Q

How can we determine how tightly R binds L?

A

By comparing the percentage of RL with the concentration of added L

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6
Q

How does an estrogen receptor recognize estradiol and not testosterone (very similar structure)?

A

The receptor has a narrow pocket lined with hydrophobic residues that fit neatly around estrogen ligands (but sterically clash w/ some other ligands like testosterone)

Proteins (receptors) can selectively bind certain molecules.
- have binding pockets that are complementary to ligand shapes

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7
Q

Does the strength of binding change with ligands that are mirror images of each other?

A

Yes, 2 estrogen ligands that are mirror images of each other showed a difference in L1/2 values.

Subtle features of ligands can be recognized

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8
Q

Why is receptor ligand binding a dynamic process?

A

Receptors and ligands are constantly associating and disocciating.
- at equilibrium and L1/2, each receptor is constantly releasing and rebinding ligands

at equilibrium, the dissociation and association rates must balance

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9
Q

Fractional saturation (Y)

A

The fraction of all possible binding sites that contain bound oxygen
- 0 (all sites empty) to 1 (all sites filled)

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10
Q

Oxygen binding curve

A

Plots the fractional saturation vs the concentration of oxygen

concentration measured by its partial pressure (pO2) in torrs

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11
Q

Metmyoglobin

A

Myoglobin bound to iron in the Fe3+ (ferric) state

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12
Q

Myoglobin’s oxygen binding curve

A

Hyperbolix (fractional saturation increases quickly, then plateaus)

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13
Q

P50 on for oxygen-binding curves

A

Half saturation of the binding site (analagous to L1/2)

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14
Q

When does P50 occur for human myoglobin?

A

at 2 torrs –> myoglobin has high affinity for oxygen

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15
Q

What makes up the oxygen binding site in myoglobin?

A

Heme

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16
Q

L1/2 for estrodial binding to estrogen receptors?

A

L1/2 = 1 nM or 10^-9 M

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17
Q

What does a heme group consist of?

A

A central iron atom and protoporphyrin IX (4 linked pyrrole rings)

iron is in the center of the 4 rings binded to N on each ring

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18
Q

In the heme group, what form must the iron be in to bind oxygen?

A

In the ferrous (Fe2+) form

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19
Q

How many bonds can iron form (in a heme)

A

6 bonds
- 4 bonds with the 4 pyrrole rings on protoporphyrin
- 2 bonds on sites known as the fifth coordination site and sixth coordination site

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20
Q

Why does iron have to be in the ferrous Fe2+ form to bind oxygen?

A

Because once oxygen binds, the EN of O will pull an electron away from Fe2+ –> Fe3+ becomes smaller and can fit into the protoporphyrin ring.

there is partial transfer of an electron from iron to oxygen

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21
Q

What are the functions of the fifth and sixth coordination sites for heme?

A
  • fifth: occupied by imidazole ring of histidine residue from proximal histidine protein
  • sixth: binds oxygen
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22
Q

Deoxymyoglobin

A

myoglobin in its oxygen-free form
- iron is in the (ferrous) Fe2+ oxidation state and the sixth site is unoccupied
- iron also lies slightly outside of the plane of the porphyrin

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23
Q

Oxymyoglobin

A

myoglobin in it’s oxygen-bound form
- once oxygen binds, the EN of it pulls electron from iron –> Fe2+ becomes Fe3+ –> iron becomes smaller and can fit into protoporphyrin ring

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24
Q

Why must oxygen be released as dioxygen and not as a superoxide ion?

A
  • superoxide is very reactive and can be damaging to biological materials
  • it would leave iron in the ferric (Fe3+) state where it can’t bind oxygen again
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25
What helps prevent the release of superoxide ion?
The binding pocket of myoglobin includes a distal histidine which donates a hydrogen bond to the oxygen molecule in order to stabilize it - hydrogen bond is from imidazole group of distal histidine
26
What molecule is a potential competitor for the oxygen-binding site on myoglobin?
Carbon monoxide (CO)
27
How does myoglobin discriminate between binding oxygen and carbon monoxide?
Studies suggest that myoglobin have evolved to have enhanced recognition of oxygen but is limited since iron has a much higher affinity for CO.
28
Structure of human hemoglobin
4 myoglobin-like subunits (each subunit has a set of α helices in the same arrangement as myoglobin) - 2 identical α chains - 2 identical β chains | hemoglobin tetramer = a homodimer of heterodimers
29
What is the structural motif of hemoglobin called?
A globin fold
30
What kind of oxygen-binding curve does hemoglobin exhibit?
A sigmoidal curve - oxygen binding at one site (within the tetramer) increases the likelihood of oxygen binding/releasing at another site
31
Why does hemoglobin bind oxygen **cooperatively**?
It allows hemoglobin to bind oxygen in the lungs (where it is plentiful) and then release oxygen in the tissues (where it is scarce) | binding rxns in hemoglobin at diff. sites are not independent
32
Compare the P50 values for hemoglobin vs myoglobin when binding oxygen.
- hemoglobin P50 = 26 torr - myoglobin P50 = 2 torr Oxygen binding affinity is lower for hemoglobin than myoglobin
33
T (tense) vs R (relaxed) states of hemoglobin
- T: quarternary structure of deoxyhemoglobin - R: quaternary structure of oxygemoglobin | deoxyhemoglobin = tense so it can't bind to O well
34
How does oxygen binding change the quaternary structure of hemoglobin?
- In R state, oxygen binding sites are not strained = capable of binding O with higher affinity - O binding to one site increases binding affinity of other sites by triggering shift from T state to the R state
35
How much does the hemoglobin α1β1 dimer rotate when oxygen binds?
15 degrees relative to the α2β2 dimer
36
What models were created to explain hemoglobin cooperativity?
- Concerted model - Sequential model
37
# Hemoglobin cooperativity model Concerted model
- the overall assembly of hemoglobin can exist only in the T or R state - binding of ligands shifts equilibrium between these 2 states
38
T-to-R transition on a hemoglobin oxygen-binding curve (concerted model)
The R-state and T-state binding curve combine to create a sigmoidal curve | Figure 3.21
39
# Hemoglobin cooperativity model Sequential model
The binding of a ligand to one site increases the binding affinity in neighboring site without inducing state conversion (T to R)
40
How does hemoglobin's behavior concerted and sequential?
- Concerted: hemoglobin w/ 3 sites occupied is almost always in the R state - Sequential: hemoglobin w/ only 1 site occupied remains primarily in the T state | Neither model fully accounts for hemoglobin's behavior
41
What movement occurs when oxygen binds to hemoglobin?
- proximal histidine (member of α helix) moves with iron ion from outside of porphyrin plane to inside - structural transition of iron ion in one subunit is directly transmitted to other subunits = communication for cooperative binding
42
2,3-Bisphosphoglycerate
Highly anionic (-) molecule that is crucial in determining oxygen affinity of hemoglobin - found in RBCs at the same concentration as hemoglobin and lowers oxygen affinity of hemoglobin ## Footnote (-) charge reduces charge aggregation between the 2 (+) Beta side chains of hemoglobin
43
How does 2,3-Bisphosphoglycerate lower hemoglobin's oxygen binding affinity?
It stabilizes the T state of hemoglobin and facilitates oxygen release - binds to pocket in hemoglobin that is only found when hemoglobin is in T state ## Footnote Without 2,3-Bisphosphoglycerate, hemoglobin would be an inefficient oxygen transporter, releasing only 8% of O in tissues.
44
What kind of effector is 2,3-Bisphosphoglycerate?
Allosteric effector - affects the binding of another molecule from a distance
45
Where does 2,3-Bisphosphoglycerate bind in hemoglobin?
In the T state aka deoxyhemoglobin, it binds in the central cavity - doesn't bind to oxyhemoglobin bc the shift obscures the BPG binding site ## Footnote Remember there's a hole in the middle of deoxyhemoglobin, which is gone after 15 degree rotation into oxyhemoglobin form.
46
Oxygen affinity of fetal hemoglobin
Fetal hemoglobin has a higher affinity for oxygen bc it has a lower affinity for 2,3-BPG - diff. in affinity allows O to effectively be transported from maternal to fetal RBCs
47
Effect of hydrogen ions and carbon dioxide on hemoglobin
Are allosteric effectors of hemoglobin (bind to sites that are distinct from oxygen binding sites) - promote oxygen release | AKA Bohr effect
48
Bohr effect
regulation of oxygen binding by carbon dioxide and hydrogen ions - hemoglobin releases more O where CO2 and H+ are higher in concentration (ex. contracting muscles) | Contracting muscles release a lot of CO2 and H+
49
What occurs to hemoglobin at a lower pH? | What is the effect of H+ ions?
Ionic bonds form and stabilize the T state = greater tendency to release O | presence of H+ ions lower pH
50
How does carbon dioxide stimulate oxygen release?
- reacts w/ water --> bicarbonate ion + hydrogen ion = drop in pH - direct chemical interaction between CO2 and hemoglobin = stimulates oxygen release
51
How does carbon dioxide stabilize deoxyhemoglobin?
It reacts with the terminal amino group = forms (-) carbamate groups - (-) charged carbamate groups participate in ionic interactions that stabilize the T state --> favors oxygen release
52
Potential treatment for carbon monoxide poisoning
Mutant neuroglobin protein (Ngb-H64Q) binds carbon monoxide 500x more tightly than hemoglobin does. | CO can bind to oxygen sites in myoglobin + hemoglobin wi/ high affinity ## Footnote In trials, CO transfer was rapid (less than 1 min) when carboxyhemoglobin was mixed w/ Ngb-H64Q
53
Example of an ionic interaction that stabilizes the T state.
- β chain C-terminal histidine has ionic interactions that are responsible for the pH sensitivity of hemoglobin. - ion pairs at the interface btwn α1β2 or α2β1 stabilize T state
53
Sickle-cell anemia
Genetic disease caused by a mutation resulting in the substitution of valine for glutamate at position 6 of hemoglobin's β chains | single amino acid substitution is responsible for disease
53
What is the mutated form of hemoglobin (in sickle-cell anemia) called?
hemoglobin S (HbS) - both alleles of hemoglobin β chain are mutated
54
# Sickle cell anemia What happens to hemoglobin due to the single amino acid substitution?
The solubility of deoxyhemoglobin is substantially decreased --> aggregation occurs - no impact on oxyhemoglobin
55
# Sickle cell anemia Why does aggregation not occur in oxygenated Hemoglobin S?
- In deoxyhemoglobin (T state), the substituted valine is on the surface and forms hydrophobic patches with phenylalanine 85 and leucine 88 (on another T-state HbS molecule) - In oxyhemoglobin, the phenylalanine and leucine are buried = no interactions
56
Sickle-cell anemia hemoglobin fibers
Hemoglobin fibers of sickled RBCs form large fibrous aggregates - fibers distort RBCs --> adhere to walls of blood vessels = block capilarries and impair blood flow
57
Toll-like receptors (TLRs)
Proteins on the outer surface of cells that recognize specific classes of molecules present in many pathogens (but absent in host) ## Footnote - part of innate immune system - extracellular domain composed largely of leucine-rich repeats (LRRs)
58
Structure of toll-like receptors
C-shaped 3D structure - extracellular domain = imperfectly repeated seq of leucine (leucine rich repeats, 20-30 residues where 6 are leucine) - have domains that anchor protein to cell surface + participate in signaling
59
What does each toll-like receptor target?
Pathogen-associated molecular pattern (PAMP) - specific molecule class targeted by TLR ## Footnote ex. TLR3 recongizes its PAMP, double stranded RNA (found in many pathogens)
60
The adaptive immune system
targets specific pathogens, even those it has never encountered - composed of humoral and cellular immune responses
61
How does the adaptive immune system recognize basically any foreign molecule?
Immunoglobulins (IgGs) have a hypervariable region - odds are that one IgG has a surface that can recognize the molecule | The rest of the IgG only has minor variations
62
Structure of immunoglobulin G | IgG = major antibody in serum
- 12 structural domains that adopt the immunoglobin fold structure - hypervariable loops (complementarity-determining regions) - 3 loops - N terminus and C terminus are at opposite ends --> structural domains strung together to form chains
63
What is the immunoglobulin fold?
Consists of a pair of β sheets that are linked by a disulfide bond - hydrophobic core in the middle
64
Light chains and heavy chains of immunoglobulin G
- 2 light chains with 2 immunoglobulin domains - 2 heavy chains with 4 immunoglobulin domains | light + heavy chains linked by disulfide bonds
65
How can Immunoglobulin G be cleaved?
Can be cleaved into 3 fragments by the proteolytic enzyme **papain**
66
What are the fragments that form when IgG is cleaved?
- 2 Fab fragments: retain antigen-binding ability - 1 Fc fragment: does not bind antigen | Figure 3.40 ## Footnote Fab = 2 "arms" of IgG (CDRs) - contains L and H chain Fc = contains only H chain
67
What is responsible for the high antibody-antigen affinity?
Numerous hydrogen bonds and van der Waals interactions contribute to the high affinity - Immunoglobulin domain changes little on binding
67
Why does an IgG have more than one Fab arm?
Many pathogens have repeating structures, so each Fab arm can stick to a diff. part of the structural repeat = IgG can stick better and have longer dissociation time
68
Cell mediated immunity
Evolved in response to the damaging properties of intracellular pathogens - use MHC class 1 proteins (embedded in cell membrane)
69
How do peptides bind to MHC 1 proteins?
MHC proteins have a deep groove that can bind peptides
70
What recurring feature is seen on the peptides that MHC class 1 proteins present?
- leucine in the 2nd position, valine in the last position - presented peptides almost always have these 2 **anchor residues** (crucial to bind to MHC)
71
T cell receptor structure
- 2 chains with immunoglobulin folds and hypervariable loops - closely related to the Fab portion of antibody ## Footnote TCR recognizes MHC peptide complex analogous to antibody-antigen complex
72
Structure of heme
- planar group - polar propionate groups at the surface, nonpolar groups buried - central iron atom in Fe2+ state - 6 coordination/binding sites
73
Why is heme not a good oxygen carrier on it's own?
It will oxidize Fe2+ to Fe3+ (can't bind O) - must be apart of a larger protein which sterically hinders the interactions between heme and O that leads to oxidation ## Footnote heme-O2-heme intermediate oxidizes O2 but protein interaction sterically hinders that intermediate formation

Chem 474 Biochemistry l (4 decks)

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