Lecture 2: Allostery

Question 1

What is allosteric regulation?

Answer 1

Non-covalent interactions which can activate or inhibit protein function.

Question 2

How do Langmuir plots work? Write out the equation

Answer 2

• Langmuir plots directly show substrate concentration vs fractional saturation.
• They are also known as direct plots.
• If P has more than 1 binding site, dissociation constants may change to cause positive or negative cooperativity.
• Positive cooperativity can allow for rapid changes in response to small changes in ligand concentration.
• At n/2, [S] will equal Kd.

Fractional saturation = n[S]/Kd+[S]

Question 3

What are Hill plots? Write out the equation.

Answer 3

We use the hill equation.

• Where h is the Hill coefficient.
• For independent binding h=1.
• Positive has h>1, with perfective positive cooperativity having h = n.
• In practice, Hill plots are not linear because h varies with ligand/protein ratio.
• At high and low S concs h = 1. The value of h at 50% saturation is a measure of cooperativity.
• In the example of Hb, we can see the oxygen affinity of the R and T states as well as the average.
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Question 4

How do the T state, R state, effectors, activators and inhibitors work? What are K and V systems?

Answer 4

• A protein can either exist in a tense (T) or relaxed (R) state.
• T has a lower affinity, R has a higher affinity.
• Activators favour R and inhibitors favour T.
• They interconvert based on effectors, which affects the equilibrium.
• Effectors can either by homotropic, when they are interactions of the same ligands (e.g. oxygen in Hb) or heterotropic, when different ligands interact (e.g. protons affecting Hb).
• K systems are when Km changes.
• V systems are when Vmax changes.

Question 5

What is the symmetry model?

Answer 5

Also known as the Monod-Wyman-Changeux model.

• When the protein changes state, molecular symmetry is conserved.
• The proteins are oligomers in which the subunits are equivalent and are related by symmetry operations.
• There is a special equation which we use for the MWC equation. We assume two states T and R.
• All 4 subunits are in the same conformation. You can’t for example, get 1 R and 3 T.
• MWC model says that allostery affects quaternary structure. It won’t affect it’s own subunit.
• The big equation can be used to find the fraction of the protein which is bound to ligand.
• When c=1 and L is small, the faction is linked the Langmuir equation.
• When c is small and L is large, the system approached perfect cooperativity.
• You can describe cooperative curves. Gives you a better idea of what is going on.
• Cooperativity is always homotropic.
• The other model requires 4 parameters.
• You can’t explain negative cooperativity with it. We can’t really explain it physiologically either.

Question 6

What is the sequential model?

Answer 6

• It states that there is a sequential change from T to R state.
• There is no assumption of symmetry.
• With no ligand, there is only T state.
• As more ligand binds, conformations change to make subunit binding more or less difficult.
• Tertiary structure changes instead of quaternary structure.
• It can explain negative homotropic co-operativity, unlike the symmetry model.
• Negative co-operativity can be seen in GAPDH for example. The advantages of it are not understood.

Question 7

What is PFK?

Answer 7

PFK is the most important control part in glycolysis. This is because delta G is so high, it is essentially irreversible.

• PFK is a homotetramer in bacteria and animals and an octamer in yeast.
• F6P is turned into F-1,6-BP.
• AMP (ADP) and F-2,6-BP are activators while citrate, ATP, and protons are inhibitors. PFK is highly cooperative (n = 3.8) with respect to F6P. ATP stimulates reaction at first but inhibits it at high concentration.
• There is no change in Vmax between the states. It is a K system, due to a change in affinities.

Two different conformations are seen.

1. A closed state (T), where a magnesium ion bridges the phosphoryl groups of the ADP and F-1,6-BP.
2. An open state when the ion only binds ADP as the products are further apart. The CD dimer rotates 7 degrees when moving from T to R. There are also changes in beta sheets like interactions at the dimer interface (at A/D and B/C).

• ADP and G6P are bound in the A subunit. ADP is at an allosteric site on the D subunit.

Question 8

What is glycogen phosphorylase?

Answer 8

Glycogen phosphorylase breaks down glycogen.

• Transition between T and R may be triggered by phosphorylation (on serine 14) or AMP binding.
• Quaternary structure change is caused by a 10 degree rotation of 1 subunit with respect to the other.
• The 280s loop becomes disordered upon activation. Oligosaccharide can then access the active site.
• 280s loop movement results causes Asp283 to be replaced with Arg569, creating a phosphate binding site.

Question 9

What is aspartate transcarbamoylase?

Answer 9

ATCase catalyses the first step in the pyrimidine biosynthesis pathway.

• It converts carbamoyl phosphate and aspartate into carbamoyl aspartate.
• It is inhibited by the final step of the pathway, cytidine triphosphate.
• It is a classic example of feedback inhibition.
• It has 12 subunits, with two trimers of catalytic subunits, and 3 dimers of regulatory subunits.
• There are large conformational changes between T and R states.
• There is a shift of 12 Angstroms. Two trimers move 12 degrees with respect to one another.
• The C1 and R4 contact is lost. The C1 and C4 contact is weakened. The catalytic site is closed by the 240s loop.

Question 10

What is the lac repressor?

Answer 10

When lactose isn’t present, lac binds to DNA and stops translation. When lactose is present, translation is started.

• LacR is a tetramer.
• DNA Kd is very low without inducer at 10^-13. Affinity for IPTG is relatively weak at 10^-6 however IPTG still controls affinity for DNA.

There are 4 subunits in a monomer:

1. N-terminal head domain with a helix-turn-helix motif.
2. Hinge region.
3. Core region has a 6-stranded sheet where the ligand binds and 4 alpha helices.
4. C-terminal helix.

• Monomers form dimers, dimers associated via C-terminal helices.
• Each dimer binds the 21 bp DNA operator sequence.
• HTH motif fits in the major groove. DNA distorts, and hinge helices fit in minor grooves.
• When IPTG binds, the T state forms, the dynamic heads of IPTG do not bind DNA.

Question 11

What is GroEL?

Answer 11

GroEL is a molecular chaperone for protein folding, working with GroES and ATP.

• It has 14 subunits arranged as 2 rings of 7, with a cavity in between. It forms a complex with GroES and 7 ATPs.
• There are cis and trans conformations.
• The GroEL has 3 flexible domains with hinges between them. The domains are apical, intermediates and equatorial.
• A hydrophobic folding cavity is created in the protein, for the actual protein to start forming.
• The unfolded protein is trapped in a cage to prevent aggregation from occurring. ATP (T) and the protein bind to the resting acceptor state.
• ATP causes changes in the bound ring so GroES can bind.
• The folding-active cis complex can then form. ATP hydrolysis allows entry of ATP and a new polypeptide into the new open cavity. A new cycle begins.

Question 12

What are GPCRs?

Answer 12

GPCRs are extensively used in signalling.

• When a ligand binds, the G-protein acts as a secondary messenger.
• In monomeric GPCRs, an allosteric molecule can alter the efficacy of the orthosteric agonist.
• In a homodimeric GPCR, binding an orthosteric agonist can alter the propensity of a second orthosteric molecule can bind. People have been trying to find drugs which targets it for years.
• Many different binding modes exist for allosteric and orthosteric ligands. Dimers or higher order ligands can form. There is a lot of possibility for allosteric control.
• Venus flytrap domain binds orthosteric agonists such as L-glutamate.
• 7TM membrane spanning region has a cysteine rich domain. This binds to allosteric modulators like PHCCC.
• The CTD and intracellular loops are responsible for trimeric G protein activation. They may represent target sites for further allosteric drugs.