Lecture 7 - Proteins in action Flashcards
Problem of oxygen availability in animals
Concentration of oxygen in saline solution is limited to approx 0.2 mmol/L, while the concentration of haemoglobin is approximately 5 mmol/L
Highly active tissue e.g. exercising muscle or the brain, is limited by the availability of oxygen
There is strong evolutionary pressure for efficient oxygen delivery
Haemoglobin
Has 4 subunits - 2 alpha and 2 beta
Each subunit has a globing (protein part) and a haem/heme (non-protein part)
Haemoglobin binding to oxygen is tightest in the lungs and releases oxygen into the tissues easier
Haemoglobin collects and binds oxygen and transports it around the body
Held together by non-covalent interactions between side chains - displays cooperatively which describes the phenomenon when one subunit affects the other
Evolved to have a weaker binding affinity to oxygen (transport) compared to myoglobin (high affinity, storage)
Myoglobin
Myoglobin, a protein found in the muscle cells of animals. It functions as an oxygen-storage unit, providing oxygen to the working muscles.
Myoglobin binds and stores oxygen for use in the muscles in bursts of high requirement
Primary structure - Approx 150 amino acids
Secondary structure - 8 alpha helices A-H and connecting loops (arranged into the globin fold)
Tertiary structure - globing fold with a hydrophobic pocket
Haem binds to His F8 (8th amino acid in helix F) in globing protein
Quaternary structure - monomeric (a single polypeptide chain)
The globin fold provides a hydrophobic pocket (Val E11 and Phe CD1) to bind a haem group
What are the two major components of a myoglobin or haemoglobin molecule?
The haem group and the globin (the polypeptide chains of the molecule)
What secondary structure dominants the globin protein?
Alpha helices with connecting loops
Active tissues and oxygen
Active tissues can use more oxygen than blood can deliver. The haem protein myoglobin meets this challenge by storing oxygen in muscles against bursts of high requirement.
Human muscles have 0.5-0.7 mmol/L myoglobin, enough for about 7 seconds of intense activity. After this store is exhausted the tissue depends on oxygen delivery from the lungs.
Whale muscles have up to 3 mmol/L, perhaps helping with deep dives.
Heme
Is a prosthetic group or cofactor
4 pyrrole rings linked together (a protoporphyrin) in a plan
6 coordinate bonds - Four to nitrogen atoms of the haem, one to a nitrogen atom of histidine F8 from the globin and one to O2
Electronic molecular orbitals of protoporphyrin give a red colour
Binding of oxygen to the Fe2+ is a reversible interaction
RIng of hydrogen and nitrogen
Histidine (N) donor from the 8th AA in F helix (HisF8). His E7 weakens binding of oxygen. HisE7 allows for weak binding so that it is reversible.
Describe the structure of the haem group
Contains a central Fe, with four pyrrole rings linked together (a protoporphyrin) in a plane
Fe2+has six coordination bond sites, what binds to each of these sites?
Four to nitrogen atoms of the haem, one to a nitrogen atom of histidine F8 from the globin and one to O2
What is the role of HisE7 in myoglobin?
Located on opposite side of His F8 and reduces binding affinity of oxygen to myoglobin, making it easier to release oxygen to the muscle cells
Spectroscopy and absorbance
Higher concentration = less transmitted light = higher absorbance
Beer-Lambert Law converts from absorbance to concentration Different wavelengths are absorbed more or less efficiently.
Shape of spectrum differs with colour and with chemical nature of solute.
Protein is colourless (but has UV absorbance).
Haem has visible absorbance (and therefore colour) that differs between bright red oxyhaemoglobin (HbO2) dull red deoxyhaemoglobin (Hb).
Myoglobin - haem interaction with oxygen
Haem Fe2+ is attached to globin protein by co-ordinate linkage to His F8. Another His in helix E (His E7) is located on opposite side of haem and
distorts binding of gas molecules to 6 th co-ordination position on haem Fe2+
This reduces the binding affinity of oxygen to myoglobin, making it easier to release oxygen to the muscle cell.
Deoxyhaemoglobin
The form resulting from when oxyhemoglobin loses its oxygen
Has a dished haem
What will weaken oxygen binding?
Anything that keeps helix F away will weaken oxygen binding
Oxyhaemoglobin
Oxygen flattens the haem, and pulls histidine F8 and helix F towards the binding site
Oxygen changes haemoglobin’s shape …
Compared to deoxyhaemoglobin, O2 binding to oxyhaemoglobin moves the Fe2+ into the plane of the haem, draws His F8 down, and repositions helix F.
Shifts in the orientation of protein secondary elements, such as helix F moving relative to helix C, are called ‘conformational changes’.
Anything that keeps the heme in the dished form (deoxy) is going to weaken oxygen binding. Anything that keeps the heme flat in the form it likes to be in when binding oxygen will make oxygen binding strong, so it will bind oxygen more strongly and this is how we tune the binding in our lungs versus out muscles to either promote binding or release oxygen
Mechanism of oxygen binding in haemoglobin
- Low PO2, oxygen has low saturation and all subunits in low-affinity T-state
- PO2 increases (i.e. in lungs) —> 1 O2 molecule binds to one sub unit, conformational change from T to R state.
- Non-covalent interactions cause the other three subunits to also change shape in response to initial subunits change —> all at high affinity R state
- Rapid binding of O2 Reverse is also true
Change from T to R can be sequential (one at a time) but mostly concerted (all at once) [realistically a mix]
Allows for better loading in the lungs and unloading in the tissues - better transporter. Haemoglobin’s tetrameter structure allows for its function as a transporter… as we increase the partial pressure of oxygen, go more into R state and therefore increase in binding of oxygen.
T state
Oxygen not bound
Taut = deoxyhaemoglobin
Low affinity - doesn’t really want to bind oxygen
R state
Oxygen is bound
Relaxed = oxyhemoglobin
High affinity - wants to bind oxygen but doesn’t want to let it go
Myoglobin and haemoglobin both show…
allosteric control’ of oxygen affinity ….LActate decreases myoglobin’s affinity for oxygen but does not bind where oxygen binds. Lactate build up in muscle promotes oxygen release from myoglobin, increasing oxygen availability for respiration
Allosteric builds on ‘steric hinderance’, the impossibility of two atoms occupying the same space
Myoglobin binds quite _____ to oxygen
Tightly
Myoglobin is O2 saturated at low pO2 only releasing O2 to muscle cells when the cellular pO2 is very low. This is shown in a ‘hyperbolic’ curve.
This suits its function as a “back-up” store of O2 in muscle cells.
Mb + O2 MbO2
The partial pressure of oxygen in lungs, or pO2, is ~100 Torr, and in resting muscle it is ~ 20 Torr.
Reversible oxygen binding but binds a lot stronger than haemoglobin
Haemoglobin oxygen binding and release
The availability of O2 to cellular proteins depends on: - The pO2 in the local environment - The binding affinity of O2 to myoglobin or haemoglobin
Haemoglobin in red blood cells in the blood needs to be able to:
- Bind O2 in the vicinity of the lungs where the pO2 is ~100 Torr
- Release the O2 in the vicinity of peripheral tissues where the pO2 is ~20 Torr
Haemoglobin therefore evolved to bind O2 less tightly.
Which one is a tetramer and which one is a monomer?
Haemoglobin is a tetramer (4 subunits) and myoglobin is a monomer (1 subunit)
Haemoglobin evolved to be a …
Tetramer - 4 globing proteins associate together non-covalently
Each globing protein contains one haem and each can bind one oxygen
