Protein function Flashcards
Interpret schematic representations of proteins to describe their quaternary structure
Yes
Describe the structural differences between myoglobin (Mb) and hemoglobin (Hb)
- Both myoglobin and haemoglobin are homologues and paralogues
- They are closely related, have the same fold and share the same fold and share sequence identity
- They exist in the same organism but carry out different but related functions
- Both proteins bind to a heme group which interacts with oxygen
- Each heme group contains an iron (II) centre which coordinates the oxygen that binds
- Myoglobin
- A monomer: a single chain/single polypeptide, single subunit that folds up into whats called a “globin” fold to make a single domain
- Stores oxygen - Haemoglobin
- Tetramer – 4 subunits each of them having the “globin” fold – has quaternary structure unlike myoglobin
- Transports oxygen
- Haemoglobin can be described as a tetramer of four subunits or, more correctly, a dimer of two “protomers”
- A “protomer” is the structural “Unit” of a protein with a quaternary structure
- The smallest unit that can be replicated by symmetry is called the protomer – so in the case of haemoglobin the protomer is the alpha-beta pair because this is the unit that is replicated by symmetry to build up the overall structure
Explain the structural basis of oxygen binding to myoglobin (Mb related to diving mammals as well)
- The two functions of Mb are:
> Storage of oxygen in muscles
> Release of oxygen when rapidly contracting muscle needs energy – when the oxygen concentration in tissue drops - Oxygen binds reversibly to the Fe2+ of the heme prosthetic group
- Mb is abundant in the muscles of diving animals, e.g. Seals, otters, and whales
Mb and diving mammals:
- Elephant seals can hold their breath for about 2 hours (they have very dark red muscles)
- When starting to dive, they slow their heart rate, stop breathing, and shunt blood to the brain, heart, and muscles
- The myoglobin concentration in muscles of diving mammals is ~10x that in humans
- Mb in elephant seals is positively charged on its surface, so the molecules repel each other and do not clump at high concentrations
- The function of many proteins involves the binding of another molecule – in the case of Mb, that molecule is oxygen
> Oxygen is called a ligand
> The ligand attaches to the binding site of the protein
Know and use the mathematical expression that describes protein-ligand binding equilibria and explain what each term represents (look at diagram)
- Equilibrium Dissociation Constant: Kd
> Preferred in Biological Sciences
> Useful in expressing ligand binding
> Represents the concentration of free ligand at which the protein is 50% saturated - Theta describes the fraction of protein binding sites occupied (ranges from 0 to 1.
- Kd tells us when half the protein binding sites are occupied; at theta=0.5 we go across the binding curve and go down, that free ligand conc will tell us what kd is for this interaction
> A low Kd (dissociation constant) indicates a strong binding interaction while a high kd suggests a weak interaction - Biotin is a high affinity/binding ligand due to its extremely low dissociation constant
Explain, using examples, the relationship between ligand binding affinity and the equilibrium dissociation constant Kd
- Biotin
- Biotin is a Vitamin – must be provided in the diet
- Biotin is needed for carboxylations
- Biotin binds the protein avidin found in raw egg white
- The Kd of 10-15 M is so small that the binding can be considered irreversible
- Biotin deficiency in humans is associated with the long-term consumption of diets rich in raw eggs.
- The biotin-avidin complex survives digestion and is lost in faeces
Wide-ranging Kd values in biology:
(Protein-Ligand-Kd value (low-high) = (high-low affinity)
- Avidin-biotin
insulin receptor (human)-insulin
- Anti-HIV immunoglobulin (human)-HIV surface protin
- Nickel-binding protein (E.coli)-Ni2+
- Calmodulin (rat)-Ca2+
Draw and interpret plots that describe ligand binding
Yes - refer to diagrams
Explain, draw and interpret the oxygen-binding curve for Hb and why Myoglobin cant transport oxygen (refer to diagram)
Myoglobin Transport Oxygen:
- Myoglobin binds oxygen very tightly
- Oxygen partial pressure in different tissues
> Lung – where oxygen is picked up by haemoglobin
> Muscle – where oxygen is delivered
- Myoglobin can’t transport oxygen in the same way as haemoglobin because it has a higher affinity for oxygen (because its an oxygen storer) and doesn’t release it readily under normal physiological conditions, unlike haemoglobin which is designed for oxygen transport
- Haemoglobin picks up oxygen efficiently in the lungs and drops off about 40% of oxygen in the tissues
- If the partial pressure of oxygen was even lower in response to the high amount of exercise, for example, it could drop off even more
> Haemoglobin can switch between different conformational states, one conformation that binds oxygen very tightly and one that binds oxygen very weakly
> Then we need a mechanism that switches between the two conformational states that is also sensitive to oxygen concentration (haemoglobin) - Hence, we get this dynamic equilibrium between these two states, the higher-affinity binding state and the low-affinity binding state
- The population of the high-affinity state depends on oxygen concentration
- As we increase oxygen conc, the population of the high-affinity state increases, at low concentrations its completely in the low-affinity state
- As the oxygen concentration of the R state (high affinity state) increases and keeps increasing as we get more and more oxygen binding
Describe the structural basis for the transition between the low- and high-affinity states of Hb
- R and T states of haemoglobin:
1. T=tense state
> More interactions, more stable
> Lower affinity for O2
2. R=relaxed state
> Fewer interactions, more flexible
> Higher affinity for O2 - O2 binding triggers a T R conformational change
- Conformational change from the T state to the R state involved breaking salt bridges between the residues at the alpha1-beta2 interface
Explain the structural basis of oxygen binding to hemoglobin (Hb)
T and R States of Haemoglobin:
- The T state is stabilised by a variety of salt bridge interactions
- Oxygen binding destabilises these interactions and allows transition to the R state
- Key interactions stabilising the T state of haemoglobin:
> Salt bridge between Histidine (HC3) and Aspartic Acid (FG1)
> HC3 means its the 3rd residue, C terminal, 2 helix H
> HC3 is the last residue in the beta subunit
> His (HC3) makes a salt bridge interaction between the positive charge on the Histidine side chain and the aspartic acid carboxylate group
> FG1 means it’s the 1st residue in the loop between helices F and G
- C terminal carboxylate group of the beta subunit that’s part of residue HC3 and the lysine residue C5 which is the 5th residue on helix C in the alpha subunit
- Oxygen binding to the nearby heme rings destabilise these contacts
- Schematic representation of salt bridging interactions that stabilise the T state
> Binding of oxygen to any one of the heme groups destabilises these interactions and favours a transition to the R state
Conformational Change is triggered by oxygen-binding
1. T state
- Helix F of the alpha subunit contributes a number of residues that interact with the iron II centre of the heme ring (e.g. histidine)
- R state
- Oxygen interacts with the Iron II and pulls it through the heme ring and flattens out the heme ring
Write the Hill equation and know what each term represents. Define cooperativity.
Cooperativity: Quantitative Description:
- Proteins that exhibit cooperative binding have multiple ligand binding sites, so our expression for the equilibrium association constant is different to that of single sided binding
Cooperative ligand binding – the Hill plot
- The slope of the Hill plot gives a measure of the degree of interaction (ie, the degree of “cooperativity”) between binding sites
- The slope of the Hill plot is called the “Hill coefficient” (nH)
- The experimentally determined slope indicates the interaction between binding sites rather than the actual number of binding sites
Explain, draw and interpret plots that illustrate cooperativity
Yes
Compare and contrast different models for cooperative binding
Two Models of Cooperativity: (for haemoglobin binding of oxygen) Concerted vs Sequential
- Concerted (all-or-none/MWC model)
- All T or All subunits R
- The whole protein switches to R or T state simultaneously
- Light (on/off)
- Transitions to R state
- No intermediates - Sequential
- Each subunit changes conformation one at a time
- Intermediates present
- Dimmer (gradual change)
- Mix of R and T states in one protein
- Induces local change
Describe the Bohr effect
Bohr effect: The Bohr effect describes hemoglobin’s lower affinity for oxygen secondary to increases in the partial pressure of carbon dioxide and/or decreased blood pH. This lower affinity, in turn, enhances the unloading of oxygen into tissues to meet the oxygen demand of the tissue.
Hb also transports H+ and CO2
- H+ and CO2 are end products of metabolism in tissues
- Hb transports about 40% of protons produced in tissue to kidneys and up to 20% of tissue CO2 to the lungs
- BUT:
- H+ and CO2 are NOT transported in the same way as O2
- H+ and CO2 do NOT compete with O2 for binding to the heme group
- In tissues: CO2 + H20 H+ + HCO3-
- So…H+ is produced both in metabolism directly, and from the conversion of CO2 to HCO3-
pH Effect on O2 Binding to Haemoglobin:
- At lower pH, the graph has shifted to the right, indicating a weaker association between oxygen and haemoglobin binding sites
- At higher pH the graph has shifted to the left, indicating greater/faster binding to oxygen and so a stronger association between oxygen and haemoglobin
- At lower pH (=higher concentration of H+), the affinity of Hb for O2 is decreased because H+ stabilises Hb in the T state
- pH is lower in tissues due to metabolism that produces organic acids (protons H+), eg lactic acid, and CO2 – the production of bicarbonate from CO2 with the production of H+ simultaneously
- The effect of H+ on lowering the affinity of Hb for O2 helps offload O2 from Hb to the tissues
- The reaction Hb + O2 HbO2 is better written:
- HHb+ + O2 HbO2 + H+
Rationalise the effect of pH on Hb affinity for O2 in terms of amino acid and protein structure
How does Haemoglobin pick up protons?
- Actively metabolising tissues generate H+, lowering the pH of the blood near the tissues relative to the lungs (catalysed by carbonic anhydrase)
Hb Affinity for oxygen depends on the pH:
- H+ binds to Hb and stabilises the T state
- Protonates His HC3, which then forms a salt bridge with Asp FG1
- Leads to the release of O2 (in the tissues)
- The pH difference between lungs and metabolic tissues increases efficiency of the O2 transport
- This is known as the Bohr effect
- Effect of H+ on the binding of O2 to Hb
- Mechanism:
> Protons (H+) are thought to bind to:
> The N-termini of the alpha subunits
> His146 (His HC3) of the beta subunit
> Other amino acids residues - In particular, the ion pair between His HC3 and ASP FGI (Asp94) stabilises the T state of Hb, causing a shift of the binding curve to the right
> The binding of O2 at 2.7 kPa decreases from ~32% at pH 7.4 to ~20% at pH 7.2
> The extra O2 released supports continued metabolic activity in the tissue
Describe how hemoglobin transports CO2
Haemoglobin and CO2 export:
- CO2 is produced by metabolism in tissues and must be exported
- 15-20% of CO2 is exported in the form of a carbamate on the amino-terminal residues of each of the polypeptide subunits
Notice:
- The formation of a carbamate yields a proton that can contribute to the Bohr effect
- This process is happening in the tissues and this release of another proton when this reaction occurs helps keep the pH low in the tissues which favours oxygen delivery
- The formation of this carbamate group on the N-terminus of each of the polypeptide chains also forms additional salt bridges because of its negative charge which also stabilises the T state and favours the release of oxygen in the tissues
- When haemoglobin reaches the lungs, this carbamate group is released destabilising the salt bridges that were formed and so favouring the R state, allowing haemoglobin to pick up oxygen more efficiently
- The carbamate forms additional salt bridges, stabilising the T state.
- Release of CO2 in the lungs favours the R state
Describe how Hb binds BPG
Effect of 2,3-BPG on the binding of O2 to Hb
- 2,3-biphosphoglycerate (BPG) is derived from an intermediate in glucose metabolism
- Like CO2 and H+, BPG interacts with Hb at a separate site to the O2-binding site and affects O2 binding
- BPG is present in red blood cells (RBCs)
- BPG is about 5 mM at sea level and 8 mM at high altitudes
2,3-Bisphosphoglycerate regulates O2 binding:
- Negative allosteric regulator of Hb function
- Present at mM concentrations in erythrocytes
- Produced from an intermediate in glycolysis
- A small negatively charged molecule, binds to the positively charged central cavity of Hb
- Stabilises the T states
Mechanism:
- BPG binds to Hb and decreases the affinity of Hb for O2:
- HbBPG + O2 HbO2 + BPG
- Hb has a single binding site for BPG at the central cavity, which is larger in the T state than in the R state
- BPG binding to residues around the cavity stabilises the T state
Explain why BPG is important for oxygen release
Yes
Explain why higher BPG concentration is advantageous at high altitude
2,3-BPG allows for O2 release in the tissues and adaptation to changes in altitude:
- Blue represents the normal oxygen binding curve for haemoglobin
- Black curve represents the binding curve of hemoglobin in the absence of BPG – shows no cooperativity and very tight binding (in the absence of BPG the R state is highly stable and T state is rarely there)
- Green curve oxygen binding curve shows that increased BPG makes hemoglobin bind oxygen more weakly
- BPG = 5mM (blue curve) – at sea level altitude
- At sea level, about 38% of O2 in saturated Hb (in the lungs) is delivered to the tissues
- Recap: saturated means the percentage of haemoglobin molecules that are bound to oxygen is high
- At high altitudes, pO2 in the lungs decreases so Hb is less saturated and O2 delivery decreases to 30% - BPG = 8mM (green curve) – at high altitude
- Affinity of Hb for O2 decreases and more O2 is delivered to the tissues (37% to 38%)
Discuss the functional effect of carbon monoxide binding to Hb
Carbon monoxide binding:
- Carbon monoxide binds at the same place as the oxygen binding site in the haemoglobin (the iron heme group)
- Carbon monoxide binds very tightly than oxygen – essentially a competitive inhibitor to oxygen binding
- Oxygen binding stabilises haemoglobin into its high-affinity R state and so does carbon monoxide
- Anemic people with only half functional Hb level of healthy people manage
- Remember Theta typically represents the fractional occupancy or proportion of occupied binding sites by a ligand
- But, if 50% of Hb subunits are bound to CO (a low-level by-product of metabolism), it can be fatal
- CO binds very strongly to Hb causing HbCO to accumulate
- CO increases the affinity of the remaining Hb subunits for O2, so an Hb molecule that binds two CO molecules can bind O2, in the lungs but can’t readily release it in the tissues
- High (dangerous) levels are largely a product of human industrial activities
Describe the quaternary structure of antibodies and visualise it. Why have antibodies evolved to be multivalent?
- Immunoglobin G (IgGs) are proteins that consist of four subunits: 2 heavy chains and 2 light chains, both involved in binding to the antigen ligand
- The variable regions at the end of each chain undergo conformational (i.e shape) change when they bind antigen
- It’s a tetramer because it contains 4 chains, but the symmetry indicates that it’s a dimer of light chain, heavy chain pairs
- Multivalent binding is more effective due to “avidity”
Describe the binding properties if myoglobin and haemoglobin.
Binding curves:
Saturation with O2 = what proportion of the binding sites of the molecule are occupied
- PO2 = partial pressure of oxygen/concentration of oxygen
As the conc of oxygen goes up, more binding sites get filled – with myoglobin they fill rapidly close to saturation
Haemoglobin’s curve is S-shaped which occurs due to its allosteric interactions
* Multimers can exhibit “allosteric” cooperative binding
Know structure of heme prosthetic group in myoglobin
Yes
Explain myoglobin binding curve (refer to diagram)
- A steep curve demonstrates tight binding; half the ligand binding sites will be occupied at quite a low conc of oxygen/at a very low partial pressure
- Recap: partial pressure is the pressure exerted by an individual gas in a mixture
> For a gaseous reaction at constant temp, conc is directly proportional to the partial pressure of a species (as the ideal gas law shows pressure is directly proportional to the number of moles/concentration when volume and temperature are constant)
