Term
| How is function of protein determined? |
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Definition
| Structure --> 3D structure determined by 1D structure. |
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Term
| What kind of protein is myoglobin vs. hemoglobin? |
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Definition
Myoglobin = monomeric (1 polypeptide + 1 heme)
Hemoglobin = Heterotetrameric (4 subunits: 2 Beta + 2 Alpha) |
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Term
| What is the difference in WHERE Hb and Mb bind O2? |
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Definition
Hb: binds O2 in lungs + release in tissue.
- extracellular + works at higher pressures
Mb: binds O2 in muscles.
- intracellular |
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Term
| What kind of fashion does O2 bind to these? |
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Definition
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Term
| What is the function of myoglobin? |
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Definition
1) Perform O2 diffusion at membranes of muscle cells.
2) Local reserve for O2 (for exercise)
3) Store O2 in aquatic animals
Note: whales have lots of myoglobin (large local reserve) |
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Term
| What does the function of proteins depend on? |
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Definition
Ability to bind other small molecules - ligands - in reversible fashion
ligand + protein = ligand-protein complex |
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Term
| How does affinity for X (ligand)and Y (protein) work? |
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Definition
| Greater the affinity of Y for X, the more XY-complex there will be at any concentration of X or Y separately. |
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Term
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Definition
Affinity level of Y for X.
Keq = [x*y]/[x][y]
Cases:
- Large Keq = higher proportion of XY to X and Y separately.
- Smaller Keq = lower proportion of XY to X and Y separately.
Keq > 1 = XY dominates
Keq < 1 = X/Y separate dominates |
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Term
| What kind of relationship exists between [ligand] and % saturation? |
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Definition
Myoglobin: Hyperbolic relationship
% protein complex increases as [ligand] increases UNTIL asymptote is reached (100% saturation) |
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Term
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Definition
The inverse of Keq:
[x][y]/[xy]
Note: Kd differs from protein to protein. |
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Term
| What does Kd say about affinity of Y for X? |
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Definition
The larger Kd --> lower affinity
The lower Kd --> higher affinity |
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Term
| What is a change in the graph when protein has a lower affinity for ligand? |
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Definition
- The Kd is a lot larger
([x][y]/ [xy] = bigger #)
- The curve is shifted downward
Note: The curve still approaches the 100% saturation |
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Term
| What are the indicators of affinity properties for [ligand] vs. % saturation graphs? |
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Definition
Higher curve = lower Kd = more affinity for ligand.
Lower curve = higher Kd = less affinity for ligand. |
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Term
| What is the structure of Myoglobin? |
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Definition
- 153 aa
- 8 a-helix + irregular structures
- 1 heme prosthetic group
Note: prosthetic groups are incorporated into 3D structure for added functionality in a protein! |
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Term
| How many helixes are in Mb and how are they ordered? |
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Definition
| There are 8 helices that are ordered by Alphabets A-H. |
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Term
| Where does heme sit in all this mess? |
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Definition
| In hydrophobic pocket between helix E and F |
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Term
| What is the shape of heme? |
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Definition
circular + planar.
Loops almost like a diamond with 4 N vertices and central Fe 2+ ion. |
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Term
| What is the structure of heme? |
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Definition
"star" porphyrin ring with a central Fe 2+ ion held by 4 Nitrogen atoms.
- 2 substituents at bottom = polar (charged) PROPIONYL groups.
- Rest substituents = non-polar aliphatic |
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Term
| Where do the polar groups on heme face? |
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Definition
| The outside surface as they are polar |
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Term
| Where does most of heme face? |
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Definition
internal side of Mb.
- interacts via hydrophobic interactions. |
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Term
| What are prosthetic groups? |
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Definition
| non-polypeptide structures added to ppeptide structures for increased functionality. |
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Term
| How are porphyrin rings in heme held in place? |
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Definition
1) Hydrophobic interactions
2) Coordination bond between Fe 2+ and "HisF8" |
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Term
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Definition
| Histidine located on the F helix and is the 8th aa down. |
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Term
| What is another name for Hisf8? |
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Definition
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Term
| What is a coordination bond? |
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Definition
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Term
| What are 2 primary roles of Hisf8? |
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Definition
1) bind heme tightly to polypeptide.
2) Prevent oxidation of Fe 2+ of heme into Fe 3+ |
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Term
| How do heme and Hisf8 interact? |
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Definition
| Via coordination bond of the N group on histidine and Fe 2+ ion of heme. |
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Term
| Which of the following serious of aa is MOST likely to be found in interior of Mb? |
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Definition
Note: interior is hydrophobic environment in all globular proteins (Mb is globular).
Hence, all serious with 100% non-polar aa would work. Ex.
-Val-Leu-Ile-Phe-Met- |
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Term
| How many coordinations positions does Fe 2+ ion in heme have? To what are they coordinated with? |
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Definition
1) 4 coordinations with Nitrogens
2) 5th coordination with proximal histidine.
3) 6th position = binding site (available) |
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Term
| Where does O2 bind to in heme group of Mb? |
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Definition
| O2 binds to the 6th coordinate position of the Fe 2+ ion. |
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Term
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Definition
| - Distal histidine (up in the tower) vs. Proximal histidine = bottom of tower. |
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Term
| What are 2 roles of the distal His? |
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Definition
1) enhance O2 binding to 6th coordinate of Fe 2+
2)Discourage CO binding |
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Term
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Definition
| - H-bond with O2 that is bound to 6th coordinate of Fe 2+ --> assist in that binding. |
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Term
| How is the oxygen binding site in Mb designed? |
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Definition
| Optimize binding specificity & affinity. |
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Term
| How does specificity & affinity relate in O2 binding site? |
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Definition
| There is a reduced affinity for CO which leads to increased specificity for O2. |
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Term
| What reduces affinity for CO? |
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Definition
Distal His takes up space that CO would require if bound to 6th coordinate of Fe 2+.
Result: unfavourable --> low affinity.
*high conc. CO needed for affinity to rise. |
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Term
| How does Mb react in different muscle requirement situations? |
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Definition
% bound reaches near 100% in resting muscles.
% bound reaches 20-30% in active muscles.
*This relation is hyperbolic + reversible |
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Term
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Definition
[ligand] when there is 50% saturation of available Mb.
If there is greater [ligand] at 50% saturation the Kd is higher and affinity = lower. |
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Term
| What is the Quaternary structure of Hb? |
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Definition
Heterotetrametic: 4 different polypeptide chains (subunits)
Subunits = 2-Alpha + 2-Beta |
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Term
| What is the similarities between the A-globins & B-globins & Myoglobin? |
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Definition
All 3 polypeptides have:
- 8 a-helices
- heme binding pocket E-F |
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Term
| What are the globins A,B,Myo- called? |
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Definition
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Term
| How do the globins A,B,Myo- differ? |
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Definition
1) polypeptide chain lengths
2) Primary sequence of aa (25% same) |
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Term
| What is the breakdown for Hb? |
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Definition
4 polypeptide chains
2 A-globins + 2 B-globins
1 heme/polypeptide = binds 4 O2/Hb |
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Term
| What is the breakdown for Mb? |
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Definition
1 polypeptide chain
1 heme - binds 1 O2/Mb |
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Term
| What is the nature of sequences in homologous proteins? |
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Definition
Similar/Identical aa in key locations in protein.
Purpose = retain functioning |
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Term
| What tends to be the extent of differences? |
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Definition
| Conservative substitutions that have minor effect on structure (Leu & Ile) and (Thr & Ser) |
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Term
| What are critical substitutions? |
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Definition
AA substitutions that affect the primary structure to change 3D structure --> change function.
Ex. Ser & Val OR Phe & Lys |
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Term
| What is the proportion difference for hydrophobic residues in Hb vs. Mb? |
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Definition
Hb = more non-polar aa since quaternary structure determined by hydrophobic effect.
Mb = monomeric so only tertiary structure -> fewer residues |
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Term
| How to A-globin & B-globin of Hb bind O2 in relation to Mb? |
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Definition
SAME
- O2 is at 6th coordinate position of Fe 2+ ion in heme ring
- Critical residues @ O2 binding site (His F8, His E7) = invariate. |
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Term
| Why are critical residues in variant? |
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Definition
Need these to maintain the 4 purposes:
1) Proximal: coordinate with Fe 2+ to prevent oxidation + bind heme tightly
2) Distal: enhance O2 binding and deter CO binding. |
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Term
| Which O2 binding relationships apply to Mb and Hb? |
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Definition
Mb = hyperbolic
Hb = sigmoidal |
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Term
| What are characteristics of a hyperbolic curve? |
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Definition
CONSTANT bonding affinity
- Ligand affinity (Kd) does not change
Ex. Mb |
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Term
| What are characteristics of a sigmoidal curve? |
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Definition
COOPERATIVE binding affinity.
- Ligand affinity (Kd) changes are more ligand binds
Ex. Hb
"easier to continue that it is to begin" - DNA melting |
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Term
| Why is sigmoidal O2 binding curve essential? |
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Definition
- The supply to tissues is satisfied by Mb because it stays O2 bound at pO2 between 20 and 40 --> not ideal.
Hb, however, enhances the % unloading of O2 between this range.
*Hb's affinity for O2 is cooperative process. |
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Term
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Definition
Similarity: reversibly bind/release O2.
Difference:
- Mb: O2 transport within tissues
- Hb: O2 transport from lungs to tissues
Sigmoidal O2 binding curve:
- Cooperative process
- Needed for efficient O2 delivery
*Result of changes in binding affinity. |
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Term
| How does Hb change its affinity for O2 (aka. how does the sigmoidal curve come about?) |
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Definition
- There are 2 states of Hb
1) Tense (T) state = low affinity
2) Relaxed (R) state = high affinity |
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Term
| Why are the states called T and R? |
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Definition
T state = more ionic interactions
R state = less ionic interactions |
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Term
| Where is His involved in this and what's the purpose? |
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Definition
Location: involved in subunit interface
Purpose: Allows transmission for changes in stricture from one subunit to the other |
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Term
| What is the difference in His residue position in Deoxy Hb vs Oxy Hb? |
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Definition
Deoxy Hb = His risidue sits with Thr and Pro
- Oxygenation occurs-
Oxy Hb = changed shape and His residue now sits with two Thr residues |
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Term
| What does oxygenation cause in Hb structure? How? |
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Definition
| Conformational change by altering positioning of His residue at the subunit interface. |
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Term
| What is the sigmoidal relationship a result of? |
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Definition
| The transitionary conformational changes from T state to R state in subunits |
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Term
| Compare aspects of T and R states of Hb: |
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Definition
Tense (T)
- Low affinity for O2
- Deoxy Hb (no bound)
- Large central cavity (more ionic interactions)
Relaxed (R)
- High affinity for O2
- Oxy Hb (bound)
- Smaller central cavity (less ionic interactions) |
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Term
| What are effectors? What do they do? |
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Definition
Compounds that, when bound to a protein, change the affinity at other binding sites.
simpler: "if you bind the effector somewhere, then it will affect the binding of something else elsewhere."
Note: bus analogy |
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Term
| What are the types of effectors? |
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Definition
Homoallosteric - binding of thing affects further binding of same thing.
Heteroallosteric: binding of thing affects further binding of DIFFERENT thing. |
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Term
| What's the difference between activators and inhibitors? |
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Definition
Activators - increase the binding affinity.
Inhibits - decrease the binding affinity |
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Term
| Where does allostery take place usually and why? |
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Definition
4D structure.
Why? - many potential binding sites. |
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Term
| What are the different combinations? |
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Definition
Homoallosteric activator - binds to INC binding of SAME thing.
Homoallosteric inhibitor - binds to DEC binding of same thing
Heteroallosteric activator - binds to INC binding of DIFF.
Heteroallosteric inhibitor- binds to DEC binding of DIFF |
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Term
| What is another name for what homoallosteric activators do? |
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Definition
Cause positive cooperativity:
- The binding of the ligand (X) at site 1 on a compound enhances the binding of SAME ligand at other 4 sites. |
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Term
| How do changes in [X] shift conformations? |
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Definition
> [X] results in shift to conformation where X IS bound. (R state)
< [X] results in shift to conformation where X is NOT bound. (T state) |
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Term
| What kind of effector is X = O2? |
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Definition
| Homoallosteric activator of Hb. |
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Term
| What are the steps of conformational change that take place when O2 binds at the 6th coordination of Fe 2+? |
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Definition
1)O2 binds
2) Binding pulls Fe to plane of Heme
3) Since Fe 2+ moves up, so does HisF8 (interacts at 5th position).
4) As HisF8 moves, so does Helix F.
5) Helix F moves, which changes the conformation of entire subunit.
6) The conformational change in 1 subunit is transmitted to adjacent via change in His position @ subunit interface |
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Term
| After O2 binds once, how do the other subunits change conformation? |
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Definition
1) The subunit interface changes in the adjacent subunit which has a chain reaction...
- Helix F moves
- HisF8 moves up
- Fe moves up into plane of heme
*Note: switch to high affinity state in other subunits occurs even BEFORE the 2nd O2 binds... |
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Term
| What does a sigmoidal relation between [X] and % bound what does this indicate about [X] |
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Definition
| That the ligand X = homoallosteric activator |
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Term
| What can we say about affinity of O2 affected by other compounds (effectors X and Y) |
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Definition
1) X causes curve to shift up.
Must be: heteroallosteric activator.
Why? enhanced affinity for O2 but is a different ligand.
2) Y causes curve to shift down.
Must be: heteroallosteric inhibitor.
Why? lowers affinity of O2 but is a different ligand. |
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Term
| What does allosteric effectors refer to? |
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Definition
Other molecules (X/Y) that may be introduced to ligand-binding systems. (like O2 to Hb)
*Influence affinities and thus change sigmoidal curve shape. |
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Term
| What are the allosteric effectors for Hb? |
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Definition
1) O2 - Homoallosteric activator
2) BPG - Heteroallosteric inhibitor
3) H+ = Heteroallosteric inhibitor. |
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Term
| What is BPG essential for? |
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Definition
Formation of T state of Hb.
recall: T state is the low affinity, Deoxy, tense, large central cavity, state. |
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Term
| What is the nature of BPG molecule? |
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Definition
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Term
| What kind of AA can interact with BPG due to these properties? |
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Definition
| - Positively charged AA like His, Arg, Lys, N-terminuses... |
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Term
| What/where does BPG bind? |
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Definition
What - deoxy Hb
Where?
- In central cavity of (T-state)
- Interaction: negatives on BPG interact with +s on proteins facing central cavity. |
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Term
| What positive proteins facing the central cavity actually DO interact with BPG? |
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Definition
1) 4 His
2) 2 Lys
3) 2 N-terms |
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Term
| Why doesnt central cavity in R-state accomodate BPG? |
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Definition
Central cavity of R-state is too small!
Result: Oxy Hb doesnt let BPG interact |
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Term
| How do H+ ions affect Hb? |
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Definition
1) Metabolism increases H+ (protons)
2) This lowers pH in blood.
3) Lowered pH causes protonation of side chains
4) Groups associated with BPG binding become protonated (more positive!)
5) Result: enhance BPG binding + reduce O2 binding
Note: Increasing electrostatic itneractions with BPG = subunit interface changes. |
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Term
| What is the name of the positive affect an abundance of H+ has on BPG binding? |
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Definition
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Term
| Why is pH of blood in lungs higher in lungs and lower in muscles? |
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Definition
The Ph needs to be relatively lower in the muscles because this is where O2 in needs to be released.
Lower pH --> more H+ --> Enhanced BPG binding --> favoured T-state (deoxy, low affinity state) |
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Term
What is the combined effects of
- O2
- BPG
- pH
...on Hb functioning? |
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Definition
1) In lungs + tissues, any given molecule of Hb can exist in one of 2 forms (T or R)
2) The proportion of Hb in either form (equilibrium state) depends on: presence of BPG, [H+ ions], and pp O2.
3)The proportion of Hb in either form (high/low affinity) determines how much O2 is bound or released.
4) @ lungs: High pp O2, high pH. R state favoured. O2 binding triggers switch to R form.
@ tissues: Low pp O2. Low pH. T state is favoured and O2 released |
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Term
| How is the functioning of a protein determined? What is the result of this? |
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Definition
ABSOLUTELY by structure
Result: AA substitutions CAN be BAD
Ex. Sickle-cell anemia, Fetal Hb |
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Term
| What is sickle-cell anemia? How does it occur? |
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Definition
Genetic disease
How? Due to critical substitution. (Glu6 replaced by Val). Polar + charged replaced by non-polar + uncharged (BAD) |
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Term
| What is the effect of sickle mutation? |
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Definition
The critical substitution replaces Glu6 with Val @ a critical location --> small hydrophobic patch exposed between E and F helices during R to T transformation.
The Val binding here = Hb molecules aggregate into long polymers.
*Single critical AA sub results in big impact on 4D structure. |
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Term
| What is the difference between Fetal and Adult Hb? |
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Definition
- Critical substitution Ser in place of His143
Result: This His residue was involved in BPG binding and now its gone...
Effect:
1) dec BPG affinity
2) inc O2 affinity
*Shifted equilibrium towards R-state under SAME external conditions |
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Term
| What is the difference between Adult Hb vs Fetal Hb sigmoidal graph? |
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Definition
Fetal Hb = shifted up relative to Adult Hb.
Note: Mb affinity is ALWAYS up to (essentially 100% R-state) |
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Term
| What are the 3 types of His and their roles in Hb function? |
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Definition
1) HisF8 (proximal)
- tight bind heme
- prevent oxidation of Fe 2+
2) HisE7 (distal)
- Assist O2 bind
- Decrease affinity of CO
(acts to increase specificity for O2)
3) 4 His in central cavity
- bind to BPG
4) Note: His also directly involved in subunit interfaces (recall: location where His sits between changes during the switch from T to R states) |
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