quaternary structure

• properties of Hb?

  • found in blood, packaged inside erythrocytes - RBC
  • carries O2
  • has 4 subunits - a tetramer
  • two types of subunits called alpha + beta - they differ in sequence
• each chain in Hb is ~24% homologous to that of myosin

  Mb and Hb have similar 3D folds - the 3D fold of myoglobin can overlay that of a subunit of haemoglobin even through their sequences are very different
• you can get the same fold in protein structure form different sequences - different sequences and similar tertiary structures
• This graph plots the fractional saturation.

  • Mb has one binding site so when one O2 binds its 100% saturated
  • Y=1 implies all 4 sites are occupied in Hb
  • at certain pO2, there's different levels off saturation of these proteins
  • p50 value same as Y=0.5 - meaning 50% saturation
  • curve shifting to the left = higher affinity

  A) myoglobin

  B) haemoglobin

  C) 100%

  D) sigmoidal

  E) hyperbolic

  F) more

  
• what does graph tell you about the p50 values?

  • Myoglobin will be 50% saturated at a lower partial pressure than haemoglobin which shows that myoglobin has a higher affinity for oxygen than Hb
  • the higher the p50 value the lower the affinity - it takes a larger concentration for the protein to reach 50% saturation

  
• how do myoglobin and haemoglobin work in synergy?

  Myoglobin stores oxygen in muscle cells and facilitates its release during periods of increased demands, while haemoglobin transports oxygen in bloodstream, releasing it at the appropriate locations.

  At low pO2 Mb can store and use oxygen when its hard for haemoglobin to use oxygen
• what type of binding curves do Mb and Hb have?

  Mb - hyperbolic

  Hb - sigmoidal
• describe this graph...?

  blue arrow - shows how much oxygen Hb can actually deliver = steepest part of the curve

  • the steepest part - Hb is responding very fast to the demand of oxygen
  • as you lower the pO2 pressure from the steep part, Hb will start release oxygen
  • in the same partial pressure change myoglobin will release very little oxygen
  • saturation is very sensitive for the middle part of sigmoid curve but less so for hyperbolic

  
• describe the binding curve for Hb?

  • lower affinity at low pO2 - allows for readily release of oxygen to surrounding tissue
  • as pO2 increases, the O2 saturation of Hb raises steeply - this initial part of curve represents the cooperative binding of O2
• describe this graph...

  • at low pO2, saturation is very sensitive for myoglobin curve but less for sigmoidal curve of Hb

  
• what results in the sigmoidal shape curve for Hb?

  cooperative binding behaviour
• how does cooperative binding make the curve get steeper?

  • as the first O2 molecule binds to Hb, it makes it easier for the next O2 to bind and so on (increases the affinity)
  • first molecule binds with a bit of difficultly
  • converse is also true for unloading oxygen - first one harder to unload
• is there cooperativity in Mb?

  no
• describe this graph - talking about cooperativity and function

  • lungs - high pO2 so Hb saturated. this blood goes to tissues where it would unload
  • tissues - low pO2 so Hb unsaturated and delivers 66% of bound O2 due to co-operative unloading (when going from lungs to tissues)
  • in contrast myoglobin being non-cooperative releases only 7%
  • rest pO2 = 40mmHg
  • exercise = 20mmHg - steepest part (blue box) copes with oxygen demand really well

  
• 3D molecular folds are very similar for alpha and beta subunits of Hb and Mb but the real difference is that Hb is a tetramer - this allows subunit interactions
• how does the quaternary structure of Hb change on O2 binding? (brief)

  • on binding the first O2, the dimer α1ß1 and α2ß2 rotates with respect to each other by 15º and moves by 0.8Å
• what are the two primary conformations of haemoglobin?

  Tense (T) and relaxed (R)
• what is the T state?

  Hb exists in the T state in the absence of oxygen. This state is quite unstable and has a lower affinity for oxygen than R state
• what is the R state?

  The relaxed state - it has a higher affinity for oxygen. Once one oxygen binds - subunit can change into relaxed state from tense state
• the binding of the first oxygen molecule promotes the transition from the T state to the R state
• what causes the slight change in shape when O2 binds to Hb?

  • Once O2 binds - size of the iron in the heme group changes slightly and relaxes back into the plane and so dimple slightly disappears. The entire heme group undergoes a more planar, relaxed structure in the R state
  • the iron relaxing back pulls the proximal histidine and the rest of the polypeptide chain with it too
  • this movement reaches alpha-beta interface and is transmitted by helix movement over to the next haem which eases movement of next Fe into the haem plane assisting second O2 to bind
• • the fraction of molecules in R states increases as oxygen binds
  • the actual proportions of R and T state decides fractional saturation
  • R state has high affinity for O2 and T state has low affinity
  • the sum of the two curve forms the S shaped curve
• what does allosteric mean?

  Allosteric refers to the regulation of a protein's activity by the binding of a molecule at a site other than the active site.
• what is an allosteric effector?

  A molecule that binds to an enzyme at a site other than the active site and affects its activity/regulates function
• whats the difference between pure Hb and Hb found in erythrocytes?

  Pure Hb binds O2 much more strongly than those found in erythrocytes as they contain BPG (an allosteric effector)

  Pure Hb has a lower p50 value - also meaning it has a higher affinity for oxygen
• what is BPG?

  2,3-biphosphoglycerate - it binds to another site on the Hb and keeps its affinity in the right range.
• what would happen to the binding curve if Hb didn't have BPG?

  Without BPG, the binding curve would shift to the left - higher affinity which would be inefficient because it would release very little oxygen
• where does BPG bind to Hb?

  At the central cavity of subunits of the deoxygenated form of Hb (T-state), its released when an O2 molecule binds
• what does BPG do?

  It controls O2 binding, as it stabilises the T-state and so weakening oxygen binding and lowering the oxygen affinity of Hb

  This means O2 is more likely to release oxygen to tissues where pO2 is low

  
• does foetal Hb have higher affinity for O2 or does adult Hb?

  foetal
• why does foetal Hb have a higher affinity for O2 than adult Hb?

  Because foetal Hb does not bind to BPG as well as maternal. In foetal Hb, BPG binding is very low and very very weak and so it can't raise the p50 value so relative to the maternal Hb, the curve lies to the left
• foetal Hb affinity for O2 being higher allows for what?

  Increased oxygen uptake in the placenta. Oxygen is able to pass from the maternal Hb to the foetal Hb
• how does BPG interact with haemoglobin to help it perform its function?

  BPG has two phosphate groups and one COOH group and so all together the charge it makes is -5. Once bound to the central cavity, the negative groups of the BPG interact electrostatically and ionically with the positive side chains in the subunit cavity - this helps stabilise the T state as it helps the BPG to be held there.
• what amino acid side chains interact with BPG in adult Hb?

  Histidine (4) and lysine (2)
• explain how the differences in structure of foetal Hb and adult Hb aids in transmission of oxygen?

  HbF has a higher affinity for oxygen compared to HbA which is mainly due to the structural differences introduced by the gamma subunits. HbF has lower affinity for BPG which contributes to the higher affinity for oxygen. This is due to two of the histidines being replaced by serine (neutral) so there's a loss of 2 positive charges and the ionic bond interaction weakens and so has a lower affinity for BPG.
• HbF having a lower affinity for BPG means that the proportion of the HbF in T state will change and not many BPG will bind to the central cavity
• when the first O2 binds, BPG is released and so affinity for oxygen increases
• how can oxygen flow from maternal Hb to foetal Hb?

  due to the fact that maternal Hb have a higher affinity for BPG, it has a lower affinity for O2 so it can unload the oxygen easier and more efficiently for the foetus to allow for development
• what other molecule affects oxygen binding properties?

  carbon dioxide - Bohr effect
• what is the Bohr effect?

  The Bohr effect is a phenomenon in which an increase in carbon dioxide or a decrease in pH (more acidic environment where there's high metabolic activity ) causes a decrease in the affinity of haemoglobin for oxygen, resulting in the release of oxygen to the tissues.

  Tissues have 0.2 unit lower pH than lungs - this gives an extra 10% release of oxygen