Lecture 8 - proteins in action continued Flashcards
T state and R state
T state = low oxygen affinity, tense subunits therefore bigger middle (during exercise you ideally want this state)
R state = High oxygen affinity, gap in the middle closes up more because of subunits getting bigger
T state pattern
Oxygen release is favoured
Increase BPG
Increased carbon dioxide
Decreased pH/ increased H+
R state pattern
Oxygen binding is favoured
Decrease BPG
Decreased carbon dioxide
Increased pH/ decreased H+
BPG
Allosteric regulator/ It is an allosteric inhibitor of oxygen binding to haemoglobin
Carries 5 negative charges
Lys and His make up a positively charged pocket for BPG to bind to ….Stabilise the T state Hb, thus remaining oxygen are released easily and prevents oxygen from rebinding once it is released
BPG can only bind to the T-state Hb because you have more open space and the BPG can’t physically fit into the R-state - when it does so it stabilises the T state, so it keeps it in the T state and keeping it here means it prevents the Hb from moving back into the R state and the T state is the low oxygen affinity state so any oxygen still bound to the Hb his way more likely to be released and used buy the muscle tissue and oxygen that has been released is less likely to rebind (so it is more available for the muscle tissue)
Favours the T (low affinity) state and its function is to improve the unloading of oxygen into the tissue
Haemoglobin and BPG
BPG binds to deoxy-Hb by electrostatic interaction. BPG stabilises Hb in the deoxy T-state, reducing oxygen affinity. BPG is produced during respiration in peripheral tissues, so promotes oxygen release where it is needed. (higher concentration in the peripheral tissues and tends to promote oxygen release when haemoglobin gets to those peripheral tissues so it depresses oxygen binding, just where you need it so that haemoglobin unloads oxygen into the brain and the muscle where you need it)
BPG by binding to the T state disfavours oxygen binding and pulls it into a state where it has a lower affinity for oxygen
Intrasubunit changes … Helix F moves away from Helix E, dished heme becomes more planar
BPG in tissues vs in the lungs
Whilst in tissues, BPG bound - T state favoured - better unloading
In lungs - high partial pressure of oxygen causes Hb to be forced into the R state - BPG ‘pops’ out of central binding pocket
Which two intersubunit changes are part of the T state to R state transitions in haemoglobin?
Helix C shifts one turn relative to Loop FG
Binding site for BPG is distorted and lost
How will a rise in pH from 6.5 to 8 influence BPG binding to haemoglobin, and why?
Weaker binding, deprotonation of histidine side chains abolishes ionic interaction with negatively charged phosphates of BPG. Hb is not only very responsive to the amount of BPG around but to the pH of blood in which the binding is occurring e.g. acidification in active muscles with lactate build up which should strengthen BPG binding and weaken oxygen binding so that more oxygen is bumped off haemoglobin in an active muscle where the pH is lower.
Cooperativity
Only arises in multimeric proteins (at least 2 subunits to cooperate with each other)
Sigmoidal curve
Cooperativity gives you sigmoidal curve for haemoglobin
When there is little oxygen, or no oxygen around, none of the 4 subunits will bind oxygen. As oxygen concentration increases the more likely oxygen is to bind to subunits. Binding to the first oxygen is hard but the structural changes the haemoglobin makes it easier for the second haemoglobin to bind so the oxygen affinity is higher in a haemoglobin with one oxygen bound than none at all
Hyperbolic curve
Depicts myoglobin at different oxygen pressures
Only one subunit with one heme which can only bind one oxygen therefore there is a different curve shape to haemoglobin. There is either oxygen bound or no oxygen bound (no intermediates)
Allosteric control
Control binding at one place by controlling the structure of the protein
Allosteric site
A site on a protein which is not the active site, to which a regulatory molecule can bind
Active site in terms of Hb is where the oxygen binds
Allosteric regulator
Regulatory molecule that binds to allosteric site
Allosteric regulation
Regulation of a molecule by binding an allosteric regulator to an allosteric site
Bohr effect
An effect where increased carbon dioxide in blood resulting in decreased pH due to increased H+ which leads to lower oxygen affinity (T state) of Hb
CO2 is an allosteric regulator by binding to the very first amino acid of each subunit, also stabilises T state like BPG.
H+ is another allosteric regulator, adding positive charges which forms ionic bonds with negatively charged side chains close by and these new bonds will lead to structural change in the subunit and it will move it from the R state closer to the T state
As partial pressure of oxygen rises, haemoglobin gives up oxygen more easily. This is because, in order for H+ ions to form haemoglobinic acid, oxyhaemoglobin must unload its oxygen.
High CO2 and H+ cause a lower affinity to oxygen. Promote oxygen release (allosteric effectors). Increase in CO2 causes a decrease in pH. H+ binds to AA chains and CO2 binds to the N terminus and has an effect on the pH. Myoglobin does not have this effect. In the vicinity of metabolising tissues, CO2 and H+ are relatively high and the partial pressure of oxygen is relatively low
Lungs favour…
Loading R … Decrease CO2 Increase O2 - wants it to be R state Decrease H+ Decrease BPG
Normal tissue favours…
Favours unloading (T state) - respiration Increase CO2 Decrease O2 Increase H+ Increase BPG
Exercise…
Increases unloading so increases T state - respiration Increases CO2 more than normal Decrease O2 more than normal Increases H+ more than normal Increases BPG more than normal
What affect do increased CO2 and low pH have on hemoglobin? (Bohr effect)
Elevated CO2 and low pH in metabolising tissue both reduce the affinity of haemoglobin for oxygen. This causes enhanced unloading of oxygen.
BPG and CO2 effect on Hb
Hb and CO2 = curve shifts a little to the left, more to T state, lowering oxygen affinity
Hb and BPG = shift even more, BPG favours T state more
Hb, BPG and CO2 = further shift, effects of these regulators is additive
Bohr effect and BPG
Allosteric effect causes rightwards shift
MWC, concerted model
ALL subunits are ALWAYS in the same configuration
Hb follows this model
Likelihood is determined by the amount of oxygen present
Structural changes aren’t large enough to move from T state to R state (structural changes still occur, doesn’t necessarily change them completely)
High oxygen the R state is highly favoured, T state strongly favoured with low oxygen
Monod, Wyman and Changeux Subunits can be in a low-activity, tense (T) or high-activity, relaxed (R) conformation. All subunits must be in the same state (either T or R). Binding each successive substrate (S) shifts equilibrium in favour of R. Inhibitors stabilise the T form. Activators stabilise the R form.
KNF, sequential model
Different subunits can be in different configurations i.e. T state and R state
Essentially says that when the oxygen binds to the Hb protein, that subunit will undergo structural and configurational changes that are so major that that subunit will be referred to as the R state subunit while the others will be slightly changed because they interact directly with the other subunit. Once one oxygen binds it is easier for the next oxygen to bind which will then make it easier for the 3rd subunit to bind
Koshland, Némethy and Filmer One substrate binding induces a T—>R conformational change in one subunit. This conformational change influences the neighbouring subunits (i.e. cooperativity), making them more likely to bind substrate. Many conformations possible. Explains negative cooperativity.
Weaker binding of oxygen
Sometimes weaker binding is better…
The first substantial adaptation to high altitude is an increase in BPG.
This reduces haemoglobin’s oxygen binding.
Rightward shift of the binding curve delivers more oxygen to the tissues.
Carbon dioxide reduces oxygen affinity
Both directly and via the lowered pH of blood
Like BPG, elevated CO2 and low pH (elevated H+) in metabolising tissues both reduce the affinity of haemoglobin for O2, known as the Bohr effect.
CO2 can bind to the extreme N- terminal amino group – H+ can protonate certain amino acid side chains – contributes to stabilising the deoxy-Hb conformation.
Oxygen binds less tightly to Hb at lower pH - better release of oxygen into the tissues
Allosteric inhibitors
Allosteric inhibitors BPG, CO2 and H+ stabilise the T-state. This unmasks cooperativity.
In absence of inhibitors, “stripped haemoglobin” is predominately in the R- state, so shows little cooperativity.
Cooperativity is the activity of one subunit is likely going to affect the other subunits.
Foetuses and oxygen
Foetal Hb has higher affintiy for oxygen. His143 is instead replaced with a Ser (both of them) (uncharged, polar therefore doesn’t carry any positive charge so it effectively removes positive charges), so pocket is less positively charged therefore BPG has less attractions which means that the Hb will spend less time in the T state which means that foetal Hb is spending more time in R state (high oxygen configuration) binds easier and doesn’t let go of it as easy which ensures that the foetus gets all of the oxygen that it needs
The positively charged His 143 is replaced by a neutral serine residue in foetal haemoglobin which means 2,3-BPG will not bind as well. This results in a higher affinity for oxygen.
Foetal haemoglobins lack an amino acid in the BPG binding site so bind BPG less well. This allows foetus to capture oxygen in the placenta.
Foetal is less positive due to serine binding therefore there is less stabilisation of the T state therefore increase affinity of oxygen therefore can steal oxygen from the maternal haemoglobin
Beta subunits of Hb
His 143
Lys 82
His 2
All amino acids of the beta subunits are positive which creates a pocket for the negative BPG
Methaemoglobin
Oxidation of haem Fe2+ to Fe3+ shifts one subunit to the R- state conformation, without oxygen bound.
Impairs function two ways:
The methaemoglobin subunit does not bind oxygen despite otherwise being in the R-state, due to the Fe 3+.
The other subunits of the tetramer are shifted to the R- state, so do not release oxygen in the tissues as they should.
Returned by cytochrome B5 reductases and NADH,
cytochrome b5 reductase regenerates haemoglobin by reducing methaemoglobin back to Fe2+ state with transfer of electrons from NADH.
In what state is the Fe-atom in methaemoglobin? Does this lead to any effect in regards to haemoglobin’s ability to bind oxygen?
Haem Fe2+ is oxidised to Fe3+ which shifts one subunit to the R-state without oxygen bound. The methaemoglobin binds oxygen less well and the other subunits of the tetramer are shifted to the R-state so they do not release oxygen to the tissues as they should.
HbM or Boston haemoglobin
Mutation of the 58th amino acid, distal His E7, of alpha chain to Tyr. Tyr displaces the 87th amino acid, proximal His F8, itself becoming the 5th Ligand, thereby stabilising the haem in the Fe3+/ferric/oxidised state.
His E7 mutation to Tyr E7 changes the environment, causing Fe2+ -> Fe3+. Haem plane moves slightly, breaking the connection of Fe-His F8 . HbM remains in T state, with low affinity for oxygen (permanent T state therefore not a very good haemoglobin)
Normal Hb vs Boston
Normal - sterically pushes on an oxygen if it came in bound to the iron atom to ensure that the binding is not too strong, ensuring a bent bond configuration and ensuring it is not too strong there won’t be enough for an oxidation reaction to take place
Boston - Distal His E7 is changed to tyrosine (this happens in the alpha subunit). His usually doesn’t form a direct covalent bond with haem iron atom but when it changes to Tyr what happens is that the OH group on the side chain of Tyr reacts very strongly with the Haem iron atom, usually wouldn’t have a direct bond with the iron atom, the tyrosine interaction pushes away the proximal histadine which would normally interact, haem ion is now able to become oxidised which means the alpha subunit can no longer bind oxygen therefore 2 out of the 4 subunits can no longer bind oxygen and cooperativity acts and the structural changes to the alpha subunits which is abnormal which will be transferred to the beta subunits as well and these structural changes will make the oxygen affinity in the beta subunits smaller so even though the mutation is only in the alpha subunit, the beta subunits will also be affected.
HbS, or sickle cell haemoglobin
Mutation of the 6th amino acid of the beta chains. The polar charged amino acid glutamate, which is located on the surface of the beta Hb subunits is changed to the non-polar, hydrophobic amino acid valine (only has hydrocarbons in its side chain)
Problem arises since there is a hydrophobic bit sticking out and a hydrophobic pocket so they can link with each other. Proteins usually want polar on surface so they can interact with the aqueous environment to make protein soluble
Sickle shape RBCs get stuck in blood capillaries
In the beta subunit, A3 (or 6th along from the N terminus) Changes from the polar glutamic acid to non-polar valine. Causes the deoxy Hb to crystallise. Hydrophobic valine interacts with hydrophobic part of alpha. Forma a chain. Provides some malaria resistance
Haemoglobin function
Oxygen binding is weakened allosterically by BPG, CO2 and low pH.
This is described as shifting the tetramer to the T-state.
When shifted thus, haemoglobin displays cooperative binding of oxygen, evident in a sigmoidal binding curve.
The R- and T-states differ in how helix F interacts with the haem and with the helix C, and spacing between H helices.
Physiological effects of haemoglobin
Oxygen affinity is tuned in pregnancy and at high altitude.
Mutations to haemoglobin impair oxygen transport.
Sickle-cell anaemia results from haemoglobin polymerisation.
Haemoglobin is under allosteric control by which molecules?
2,3-Bisphosphoglycerate (BPG), CO2 and H+
How does BPG modify the O2 binding characteristics of haemoglobin?
Binds to and stabilises deoxy-Hb in the T-state by electrostatic interaction, reducing oxygen affinity.
How is BPG displaced from haemoglobin and why does this facilitate O2 transport?
Deprotonation of histamine side chains reduces the ionic interaction with BPG and it will not be able to bind.
The protein changes shape and without BPG the affinity for oxygen increases.
BPG binding allosterically promotes the release of oxygen bound to the haemoglobin
How does shifting from the T-state to the R-state affect how the haem interacts with the protein?
Moving from T to R state, oxygen flattens the haem which causes the His F8 to move which in turn shifts the entire polypeptide chain. The change in surface to surface interaction between subunits of haemoglobin induces cooperative binding.
