Week 11 Hitchcock Lecture 4

Question 1

What do porins do?

Why are porins only good for outer membranes and not inner membranes of a cell?

Answer 1

allow passive uptake of water soluble hydrophilic molecules up to around 600 Da in size to cross the outer membrane – they form these aqueous channels from antiparallel beta-strands that form hollow cylinders called beta-barrels
-These porins mean outer membrane is less of a permeability barrier to polar and charged molecules, but recall porins are not suitable for the inner membrane as the pores would allow ion leakage and dissipate the proton motif force, and of course there is not an electrochemical gradient across the outer membrane.

Question 2

What is Passive transport/facilitated diffusion?

Answer 2

Movement of the solute with the concentration gradient (from high concentration to low concentration)

Question 3

What is Active transport?

Answer 3

Requires an energy input to move solute against the concentration gradient (from low concentration to high concentration)

Question 4

What are Primary active transporters?

Answer 4

Depends on ATP hydrolysis (or hydrolysis of some other compound) to release free energy to drive transport

Question 5

What are Secondary active transporters?

• What do secondary active transporters do?
• What are the different mechanisms used under secondary active transporters?
• What are secondary transporters usually made up from?

Answer 5

Depends on a pre-existing ion-gradient across the membrane (typically proton-motif force but can in principle be any chemiosmotic ion-gradient) to drive transport

• Secondary transporters are carrier proteins that couple the thermodynamically unfavourable flow of one species of ion or molecular up a concentration gradient to the favourable flow of a different species down a concentration gradient. Theoretically they are reversible – can work in the opposite direction if the gradient is reversed
• uniporter, symporter and antiporter
• Usually consist of a single protein with 12 TMHs

Question 6

What do uniporters transport?

Answer 6

Active uniporters transport positively charged ions and they are driven just by the delta psi component of the PMF. These ions are positively charged and so they can flow from the positive p side to the negative n side of the membrane through the uniporter driven by the electrical charge difference across the membrane, so just the membrane potential, not the delta pH

Question 7

What do symporters transport?

Answer 7

symporters solute movement against its concentration gradient is driven by a coupling ion (proton or Na+) moving down its a concentration gradient, and both the solute and the coupling ion move in the same direction. So this is driven by dissipating some of the energy in the proton or sodium motive force with the energy released by the protons or sodium ions moving down their concentration gradient is used to drive the uptake of solute, S, against its concentration gradient.

Question 8

What do antiporters transport?

• What is usually exchanged in antiporters?
• Are antiporters electroneutral or electrogenic?

Answer 8

Where a solute and a coupling ion or two solutes in this example are moved across the membrane in opposite directions. For example, we have two solutes S1 and S2, and the unfavourable uptake of S1 is driven by the solute gradient of S2 in the opposite direction. So if the cell wants to take up a nutrient S1 which will be in lower concentration outside the cell than inside and so needs active uptake, it can do so by exporting a molecule S2 that is in higher concentration in the cell relative to the outside and so moves from inside to outside down its concentration gradient. This movement of S2 is thermodynamically favourable and so releases the energy for import of S1.

• Typically used to exchange substrate/precursor with a product
• both

Question 9

What are the Jardetzky principles for active transport?

Answer 9

Principles:

• Interior cavity with a high affinity and specific binding site for substrate Z
• Energy drives conformational change in the protein and results in a relative decrease in affinity for the substrate
• ‘Alternating access model’ – transporter never has access to both sides of the membrane at the same time
• No access to the high concentration side in the high affinity state
• No access to the low concentration side in the low affinity state

Question 10

How does LacY and the rocker-switch mechanism work?

Answer 10

LacY is in the outward facing conformation and a proton binds to the carboxyl group of a specific glutamate residue on the periplasmic side of the transporter. Binding of the proton allows the lactose to bind to its binding site in the hydrophilic cavity. The proton then moves to another glutamate on the cytoplasmic side of the transporter and this proton movement across the membrane from the p side to the n side releases free energy and drives the conformational change to the inward-facing conformation – so in this conformation lactose can’t flow with its concentration gradient as access to the periplasm is blocked. The conformational change means lactose is bound less tightly and is released into the cytoplasm, followed by the proton which has moved from the p side of the membrane to the n side with its concentration gradient, and the system resets back to the original outward-facing conformation and can transport more lactose.

Question 11

What is the elevator mechanism that is used by CitS?

Answer 11

The bacterial Na+-citrate symporter CitS uses an elevator-type transport mechanism. Translocates citrate (2-hydroxy-tricarboxylic acid) in symport with two Na+ ions. A compact transport domain twists and slides up and down through the membrane while a scaffold domain is stably anchored. Movement of the transport domain relative to the scaffold domain – different example of the ‘alternating access’ mode

Question 12

What are Tripartite ATP-Independent Periplasmic (TRAP) Transporters?

• Where are they found?
• What are the substrates present?
• How does it work?

Answer 12

R

• Found in bacteria and archaea but not in eukaryotes – especially common in bacteria that inhabit marine environments
• are organic acids with a carboxylic acid of sulphonate group, which forms a salt bridge with an invariant Arginine residue in the substrate binding protein.
• The SBP binds the substrate in a cleft between two domains through a large confirmational change akin to a Venus fly-trap, so it has a Venus Fly-trap fold, resulting in it, and the TRAP transporter as a whole, having a very high affinity for its substrate, which is in the low micromolar range. The SBP also means TRAP transporters are only used for uptake