MP1: How chemicals move across membranes

Question 1

Why are membrane proteins important?

Answer 1

• They have a diverse range of roles: enzymatic, transportation, signal transduction, cell recognition, intercellular joining, attachment (ECM).
• Several diseases are linked to mutations within membrane proteins e.g., cystic fibrosis where the chloride transporter isn’t trafficked properly.
• Implicated in drug resistance
• Major drug targets

Question 2

What percentage of human genes encode membrane proteins? How many of total protein structures solved are membrane proteins?

Answer 2

20% ; 1%

Question 3

What is the general rule about the structures of single pass TM proteins?

Answer 3

They will always be an alpha helix.

Question 4

What are the 3 zones of the bilayer, and what are their thicknesses?

Answer 4

1. Water
2. Interface (10-15 angstrom)
3. Hydrophobic core (30 angstrom)

Question 5

Describe the structure of alpha helix bundles.

Answer 5

Alpha helix bundles are compact protein structures formed by the assembly of multiple alpha helices.
They are the predominant architecture for membrane proteins.
Each alpha helix is a rod-like structure that is stabilized by hydrogen bonding between the backbone amide and carbonyl groups, resulting in a characteristic right-handed twist. When two or more alpha helices come together, they can form a bundle-like structure through additional hydrogen bonding, hydrophobic interactions, and other stabilizing forces.

Question 6

What are the possible functions of membrane proteins? [REMEMBER: JET RAT]

Answer 6

Junction - connect two cells together
Enzymes - fixing to membranes localizes metabolic pathways
Transport - facilitated/active transport
Recognition - markers for cell identification
Attachment - cytoskeleton and ECM
Transduction - receptors for peptide hormones

JET RAT

Question 7

Describe the two/three state in vivo folding model.

Answer 7

Proposed by Popot and Engelman to describe the folding of alpha helix bundles.
In the two-state model, the folding process is described as a direct transition from the unfolded state to the fully folded native state, before TM insertion and helix aggregation.

The three-state model extends this, accounting for prosthetic groups and unusual peptide conformations that might not fully form simultaneously.

Question 8

Give an overview of beta-barrels (structure, commonly found, size, therapeutic use)

Answer 8

• Protein secondary structure that consist of beta-strands arranged in a cylindrical shape, creating a barrel-like structure. The beta-strands are typically arranged in an anti-parallel fashion.
• Commonly found in bacterial outer membranes (and mitochondria/chloroplasts)
• Size varies from 8-36 beta strands
• Target of many antibiotics

Question 9

How is it thermodynamically possible for some membrane proteins to have charged residues?

Answer 9

Ensuring overall hydrophobicity is favorable.
- interact with charged lipids e.g., phosphatidylserine
- position charged residues to have interactions with other polar molecules or exposed to the aqueous environment

Question 10

How can TM helices be predicted from an amino acid sequence?

Answer 10

Hydrophobicity scales can be used to assign a hydrophobicity value to each amino acid and thus predict TM helices.
This is achieved via the sliding window method: 20 residues are taken (occupies 30Å in length which is the width of the hydrophobic core of the bilayer) and you calculate the mean hydrophobicity is within this window. The window then moves across the protein sequence to give rise to a prediction.

Question 11

What is the mean length of hydrophobic TM helices? What does this show?

Answer 11

25 residues (~37.5 angstroms)

The hydrophobic core is only 30 angstroms, so the helices must be at an angle.

Question 12

What is a tryptophan/tyrosine ‘collar’?

Answer 12

Often membrane proteins have a collar of Trp or Tyr at the interface – this can be rationalized because these are quite hydrophobic residues, but they also have something that can make hydrogen bonds. In the interface region there are lots of phosphate groups that are keen to make these hydrogen bonds and so by having these amino acids you can overcome the potential imbalance of enthalpy through the potential to make hydrogen bonds.

[Was identified in HIV glycoprotein and was thought to show how these Trp-rich regions can bind to zwitterionic membranes in a ‘velcro-like’ manner.]

Question 13

Why isn’t phenylalanine found in collars? Where do it prefer to reside instead?

Answer 13

It can’t make hydrogen bonds, so instead prefers to sit in the middle of the bilayer. This is because there’s less density in the center.

Question 14

What is lysine snorkeling?

Answer 14

Lysine’s positive end (-NH3) will often snake up to the head groups of the phospholipids to make salt bridges, via snorkeling.

Question 15

What is often the role of proline in membrane protein helices? Give an example of where proline is important for membrane protein function.

Answer 15

Many helices will also have kinks or distortions in their structures created by the presence of proline residues.

E.g., Lac permease. 7 of the 12 TM helices contain a proline and this is key to its function in helping to move the helix for conformational changes. This is common to many other membrane proteins and studies have shown that proline may have roles in channel and receptor activation.

Proline cannot make hydrogen bonds which leads to the kink, flexible enough that can cause some conformational changes. Bacteria tend to replace prolines with glycines when their proteins need a kink.

Question 16

What is the main role of phosphatidylcholine and in which part of the membrane is it most commonly found?

Answer 16

Most abundant lipid in the membrane.
- zwitterion so can form tight and stable packing with other phospholipids
- involved in signal transduction and lipid metabolism

Found in the outer leaflet.

Question 17

What is the main role of sphingomyelin and in which part of the membrane is it most commonly found?

Answer 17

• Forms lipid rafts which are involved in signal transduction and membrane trafficking
• Regulates some membrane protein functions

Found in the outer leaflet.

Question 18

Why can expressing membrane proteins for studies be difficult? How can this be overcome?

Answer 18

• Overexpression of a mammalian channel in a vector can be lethal, but studies require at least 1mg of the protein.
• Hard to purify from natural sources.
• Overcome by using bacterial homologues and eukaryotic expression systems.

Question 19

What is the problem with using detergents to purify membrane proteins? How will the method used determine structural studies?

Answer 19

They can denature the proteins, so there’s a need for non-denaturing detergents e.g., DDM and OG. However, these can be very expensive and require trial-and-error to find the right one.

Solvation strategy will impact which method is used to solve the structure.

Question 20

What is the problem with using detergents to purify membrane proteins? How will the method used determine structural studies?

Answer 20

They can denature the proteins, so there’s a need for non-denaturing detergents e.g., DDM and OG. However, these can be very expensive and require trial-and-error to find the right one.

Solvation strategy will impact which method is used to solve the structure.

Question 21

What are the main properties of cholesterol and in which part of the membrane is it most commonly found?

Answer 21

• Sterol, so has a rigid and planar structure, allowing it to interact with FA chains of other lipids which increases membrane packing and reduces fluidity.
• amphipathic (can lie in membrane)
• interacts with membrane proteins, impacting their activity and regulation

Mostly found on the outer leaflet.

Question 22

What are the main functions of PIP2 and in which part of the membrane is it most commonly found?

Answer 22

• Signaling (cleaved by PLC to give IP3 and DAG)
• Ion channel regulation

Mostly in the inner leaflet.

Question 23

What is lipid cubic phase solubilization?

Answer 23

Lipid cubic phase solubilization is a technique used for the solubilization and crystallization of membrane proteins.

The lipid cubic phase is formed by mixing a lipid monomer, such as monoolein, with an aqueous protein solution. The lipid monomer self-assembles into a highly ordered and viscous phase, with a network of internal channels and pores that provide a hydrophobic environment for membrane protein insertion and stabilization. The resulting lipid cubic phase can then be used for the crystallization of membrane proteins by introducing a protein solution into the phase and incubating it under controlled conditions to promote crystal growth.

Question 24

What are the main alternatives to using detergents to extract membrane proteins?

Answer 24

1. Amphipols: small amphiphilic molecules that can bind membrane proteins and stabilize them in solution while preserving native structure and function.
2. Lipid nanodiscs: self-assembled lipid bilayers that are stabilized by a membrane scaffold protein. Membrane proteins can be incorporated into these without the use of detergents.
3. Liposomes: artificial lipid bilayer vesicles that can be disrupted to release the proteins.
4. Cell-free expression systems

Question 25

Why are membrane proteins hard to crystallize and how can this be overcome?

Answer 25

- easily destabilized in aqueous solutions - extramembrane domains can be very flexible, preventing formation of ordered crystals - low concentrations in biological membranes 1. Stabilization of the protein e.g., lipidic cubic phase 2. Mutagenesis to stabilize the protein/facilitate crystal packing 3. Crystallization screening to find suitable conditions for crystal formation 4. Single particle cryo-EM 5. Use of antibodies to make the protein water soluble

Question 26

Why was single particle cryo-EM so important for studying membrane proteins?

Answer 26

Can solve protein structures without crystallization. Instead, samples are rapidly frozen, preserving the native state of the protein and minimizing radiation damage.

Question 27

Can NMR be used to solve structures of membrane proteins? What are the disadvantages?

Answer 27

Yes, it can provide structural and dynamic information. However, the large size and hydrophobic nature of the proteins means they pose challenges, such as signal broadening and poor solubility. Extraction techniques can also interfere with NMR.

Question 28

Why is molecular dynamics important for studying membrane proteins?

Answer 28

Molecular dynamics (MD) is an important tool for studying membrane proteins because it allows researchers to simulate the behavior of proteins in a lipid bilayer over time. MD simulations can provide detailed information on protein-lipid interactions, protein dynamics, and conformational changes that are difficult to obtain experimentally.

Question 29

How can mass spectrometry be used to study membrane proteins? What are the 3 main approaches used?

Answer 29

MS can be used to identify and quantify membrane proteins, determine post-translational modifications, and investigate protein-protein and protein-lipid interactions. One of the main advantages of MS is its high sensitivity and specificity, which allows for the detection of low abundance proteins or modifications. 1. Shotgun proteomics (PTMs) 2. Chemical cross-linking (protein-protein interactions) 3. Native MS (protein-lipid interactions)

Question 30

What are the 3 main 'regions' of protein channels?

Answer 30

Pore (P) Filter (F) Gate (G)

Question 31

Describe the overall structure of potassium channels.

Answer 31

- conserved core topology: 2 alpha helices and a linker - TVGYG filter motif with carbonyls pointing inwards for selection of potassium ions - forms a tetramer pore - large central cavity that contains water

Question 32

What is the structural difference between voltage-gated and non-voltage potassium channels?

Answer 32

VG channels have the same central pore, but each subunit has 6 helices. The additional 4 helices are what senses the voltage.

Question 33

Describe the filter and permeation used in potassium channels. How did MD play a key role in disproving crystal structures of the filter?

Answer 33

- TVGYG coordinates ions via carbonyl oxygens. - crystal structures show ions only in these positions 50% of the time, suggesting an alternating mechanism between ions and water - MD disproved this, showing no alternations (direct coulombic knock-on model) Still being argued which model is correct.

Question 34

What is the direct coulombic knock-on model of potassium channel selectivity? What is the evidence for and against this model?

Answer 34

The direct coulombic knock-on model proposes that the potassium ion is initially coordinated by a single carbonyl oxygen atom, which is then replaced by a neighboring oxygen atom through a "knock-on" mechanism. In other words, the incoming potassium ion interacts with the first oxygen atom, which then pushes the neighboring oxygen atom out of its coordination site, allowing the potassium ion to occupy it. This process repeats itself until the ion reaches the other end of the filter. For: - XRC - MD simulation Against: - 'snug-fit' model - 'field ionization' model

Question 35

How is selectivity achieved in potassium channels?

Answer 35

The Toy model: there's a balance between favorable electrostatics and unfavorable repulsion that only balances sufficiently for potassium ions, but not sodium ions. Sodium ions are too small so the repulsion term is much greater. Larger ions cannot fit through the pore.

Question 36

How was the initial model for potassium ion channel selectivity disproved?

Answer 36

It was initially suggested that specific interactions compensated for potassium ion dehydration, but these interactions weren't good enough for sodium. Against: - XRC resolution used was too low - VDW radii of the O are too close to allow K to pass, so there must be some movement - Hydrogen bonds aren't rigid - TVGYG can exist as TVGFG and still show selectivity, despite F lacking hydrogen bonds - Large thermal movements observed

Question 37

How is gating achieved in potassium channels?

Answer 37

Glycine hinges in bacteria play a key role on allowing the inner helices to bend ‘out’ and enable channel opening. In mammalian voltage-gated channel there’s a more elaborate motif: Pro-X-Pro. This gives mammalian channels their gating ability.

Question 38

How is voltage-sensing achieved in VGK channels?

Answer 38

Voltage-gated potassium channels contain 4 additional helices on their N-termini to sense voltage. The S4 helix contains a large number of arginines, identified via SDM mutagenesis. By creating a chimeric structure between two types of Kv channels, you could see S4 helices interacting with the lipid bilayer. Current model: -70mV (resting) puts the S4 helix in a ‘down’ position. As the potential becomes more positive, it repels the positive charges in the S4 voltage sensor up, dragging the S4/S5 linker and allows the S6 helix to then move to an open state. This is facilitated by a key Phe residue on the S3 helix, acting as a ‘hydrophobic gasket’ to hold the whole thing together.

Question 39

Describe the overall structure of sodium channels, including the selectivity and plug regions.

Answer 39

Consist of large alpha subunits that associate with other subunits. An alpha subunit forms the core of the channels and is functional on its own. The alpha subunit has 4 repeat domains, each containing 6 TM helices (much like VGK channels). Selectivity region - formed by P-loops, is the narrowest region and responsible for ion selectivity. Plug region - linker between S5 and S6 forms a plug when the channel is inactivated.

Question 40

How is selectivity achieved in sodium channels?

Answer 40

These doesn’t have the carbonyl oxygens pointing inwards, and instead the selectivity is achieved through partial dehydration: sodium ions will only use some of its water molecules via glutamate side chains. This is because the protein cannot compensate for losing all of its water molecules.

Question 41

What are the substrates, mechanism, roles, and locations of P-type ATPases?

Answer 41

Ions Alternating access Ion homeostasis, acidification, apoptosis Plasma membrane, ER/SR membrane

Question 42

What are the substrates, mechanism, roles, and locations of rotary ATPases?

Answer 42

Protons Rotary Acidification, proton-driven ATP synthesis All membranes

Question 43

What are the substrates, mechanism, roles, and locations of ABC transporters?

Answer 43

Ions, vitamins, lipids, drugs... Alternating access Nutrient import, drug export, lipid transport Plasma membrane, ER membrane

Question 44

Name the 3 types of P-type ATPases found in cardiomycocytes.

Answer 44

Na/K ATPase SERCA Plasma membrane calcium ATPase (PMCA)

Question 45

Describe the domain structure of P-type ATPases

Answer 45

3 cytosolic domains: nucleotide, phosphorylated, actuator.

Question 46

How is selectivity achieved in P-type ATPases?

Answer 46

The E1-E2 scheme is used to explain the ion selectivity of these pumps. In the E1 state, the ATP-binding site is exposed to the cytoplasmic side of the membrane, and the ion-binding site is exposed to the extracellular side of the membrane. In this state, the ion-binding site has a high affinity for the ion that is transported by the pump. For example, in the case of the Na+/K+ ATPase, the ion-binding site has a high affinity for sodium ions. Upon ATP hydrolysis, the pump undergoes a conformational change to the E2 state. In this state, the ATP-binding site is occluded, and the ion-binding site is exposed to the cytoplasmic side of the membrane. In this state, the ion-binding site has a low affinity for the ion that was previously bound, causing it to be released into the cytoplasm. In the case of the Na+/K+ ATPase, this results in the release of sodium ions into the cytoplasm. E2 is dephosphorylated to return to the E1 state.

Question 47

How is occlusion achieved by P-type ATPases?

Answer 47

When ATP binds, the N-domain moves inwards, and the A-domain rotates to form a hydrolysis site for ATP. The rotation of the A-domain causes helices 1 and 2 to move upwards slightly and shut off the intracellular entrance pathway to the calcium ion, causing occlusion. Thus, ATP binding and phosphorylation lead to calcium occlusion by domain movements.

Question 48

Describe the structure of ABC transporters. How do these change when ATP binds?

Answer 48

4 domains: 2 nucleotide-binding and 2 TM ^ conserved coupling helices link these together. NBD dimerize when ATP binds, sandwiching 2 ATPs between 2 NBDs. Once ATP is hydrolyzed, the dimers dissociate. NBD contains a RecA-type core with Walker A motifs.

Question 49

Describe the mechanism of maltose importers as an example of ABC transporters.

Answer 49

Maltose binds to a substrate-binding protein (SBP) prior to docking to the importer. Structural changes bring the two NBDs closer for dimerization once they have been loaded with ATP. Full dimerization forces the TM domains to go from the inwards to the outwards facing conformation which opens up the importer to maltose. ATP is hydrolyzed, releasing Pi to allow for the TM domains to re-enter the inwards facing state and thus release maltose and the SBP.

Question 50

How can ABC transporters, such as human P-glycoprotein, cause drug resistance?

Answer 50

e.g., human P-Glycoprotein – this is a highly promiscuous multidrug transporter that can transport hundreds of structurally unrelated hydrophobic amphipathic compounds. This is a problem for drug delivery in cancer cells when P-glyco is upregulated, conferring chemotherapy resistance to cancer cells. It’s expressed in barrier tissues such as the blood-brain barrier. It works via a flippase mechanism, binding a substrate from within the membrane to expel them. The substrate binding pocket is very large in the protein so it can even accommodate more than one substrate molecule at a time! The NBDs also don’t work in concert but alternate, leading to the alternating access shifts in conformation of the TM domain.

Question 51

Describe the structure of eukaryotic V-type ATPases.

Answer 51

These complexes are composed of two main components: the V0 domain and the V1 domain. - V0: TM proton-conducting domain, consisting of several subunits that form a ring-like structure within the membrane. - V1: ATP-hydrolyzing domain, located on the cytoplasmic side of the membrane. Subunit 'A' binds ATP and is responsible for its hydrolysis. The V0 and V1 domains are connected by a central stalk, linking the proton transport and the catalytic activity. Together, these domains form a large 'L-shaped' protein complex that spans the membrane and allows for the efficient pumping of protons across biological membranes.

Question 52

Compare eukaryotic V-type ATPases to bacterial V/A/F-type ATPases.

Answer 52

Eukaryotic V-type ATPases and bacterial V/A/F-type ATPases are both types of ATP-driven proton pumps that generate a transmembrane electrochemical gradient by transporting protons across biological membranes. Eukaryotic: - at least 14 subunits - transport protons via a rotary mechanism - functions in acidification of intracellular compartments Bacterial: - 8-11 subunits - transport is through a channel in the c-ring - functions in ATP synthesis, flagellar movement, and nutrient uptake - can do both ATP synthesis and hydrolysis - can be driven by protons or sodium ions

Question 53

What general structural rule can be applied to outer membrane vs inner membrane proteins? Why is this thought to be the case?

Answer 53

OMP: beta-barrels IMP: alpha helical Inner membrane is much simpler with phospholipids on both leaflets. OM inner leaflet also contains phospholipids, but outer leaflet contains LPS.

Question 54

Describe the energetic barrier differences across the outer membrane, and what creates them.

Answer 54

The outer membrane contains phospholipids on the inner leaflet, and LPS on the outer leaflet. The lipid A headgroups of LPS create much larger energy barriers compared to phospholipid headgroups. As such, compounds such as benzene can easily move through the phospholipid headgroups, but it then encounters an energetic barrier at the lipid A headgroups.

Question 55

What are the 3 oligomeric configurations of membrane beta-barrels? Give an example of each.

Answer 55

1. single chain making a single barrel e.g., OmpG 2. three chains making three barrels e.g., OmpF 3. seven chains making one barrel e.g., alpha-hemolysin

Question 56

Describe the structure of an average beta-barrel membrane protein.

Answer 56

- antiparallel beta strands that make hairpins - 8-24 strands - most are evenly stranded - strands are connected by short turns at the inner leaflet and longer loops at the outer leaflet (in bacteria) - high thermal stability

Question 57

Why are beta-barrels harder to predict structures for than alpha-helices?

Answer 57

There's a great deal of sequence variability within barrels, particularly so in loops. - varying numbers of strands and orientations whereas alpha helices have a characteristic right-handed structure, with a predictable backbone hydrogen bond structure.

Question 58

What are pore-forming toxins and what do they do?

Answer 58

α and β pore-forming toxins (based on the secondary structure of the membrane spanning region) are virulence factors produced by bacteria specifically to form pores in target membranes. These alter the membrane permeability of their target cells with the aim of leading to cell death.

Question 59

What are the functions of beta-barrel extracellular loops?

Answer 59

Recognition Adhesion to other cells Gating [may play a role in orienting natural substrates and specificity] Research is showing that OMPs actually form clusters in LPS-free regions which may play a role in specificity via their loops.

Question 60

What have the interiors of beta-barrels been shown to have a role in?

Answer 60

Specificity (either of large molecules or ions)

Question 61

Why does specific transport inherently plateau, unlike non-specific transport?

Answer 61

Specific transport requires the substrate to interact with some form of binding site which will inherently set a maximal rate of transport, making the rate plateau. Non-specific transport doesn’t have this interaction and so won’t have such a limit.

Question 62

What are general pore-forming toxins? Give an example.

Answer 62

A subclass of PFTs that don't require a specific target receptor on the host cell surface to enter the cell. Instead, they can bind to any membrane and form pores, allowing passive permeation of polar molecules up to 600Da. e.g., alpha-hemolysin which forms a 'mushroom-shaped' pore i.e., the stalk penetrates the membrane, whilst the cap sits on the surface.

Question 63

Describe the structure and selectivity of OmpF.

Answer 63

OmpF is a porin protein found in the outer membrane of Gram-negative bacteria, such as Escherichia coli. It forms a trimeric structure, with each monomer consisting of 16 beta-strands that form a barrel-like structure with a central pore. Loop 3 folds back into the barrel which creates an eyelet region that aids selectivity by blocking transport of larger molecules. This eyelet is also lined with negatively charged residues that repel anions while attracting cations.

Question 64

What are Occ channels? Why are they important in conferring antibiotic resistance?

Answer 64

The name "Occ" stands for "outer membrane carboxylate channels" and refers to the fact that these channels have structural similarities to both porins and cytolytic toxins. Occ channels are made up of beta-barrel structures that form channels through the bacterial outer membrane, allowing the transport of small molecules across the membrane. Occ channels allow for secretion of antibiotics, but also they are much more specific than general PFTs and so prevent most antibiotics from even getting into the bacteria.

Question 65

What is the role of the arginine ladder in Occ channels? Give an example of an Occ channel using it's ladder.

Answer 65

Occ channels are specific channels, unlike general PFTs. This is due to the presence of an arginine ladder within the channel. It's thought that only substrates with carboxyl groups can efficiently be transported, as the group is required to interact with the guanidium groups on the arginines. MD simulations were used to show how arginine moves through the OccD1 channel. It's thought that the arginine makes contact with the ladder, but must be in a specific orientation for this to work i.e., the carboxyl group must be facing down to direct the pathway.

Question 66

Give 3 examples of OMPs that have been shown to contain lateral gates via XRC.

Answer 66

1. FadL - transports long chain FA 2. CymD - transports p-cymene 3. TodX - benzene and toluene [These are all members of the FadL family of proteins.]

Question 67

How is are monoaromatic hydrocarbons, such as toluene, taken up by gram-negative bacteria?

Answer 67

Through a combined experimental and computational approach, it was shown that uptake was via lateral diffusion through FadL channels. Contrary to classical diffusion channels, TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by LPS. One study suggested that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing, and gut biology.

Question 68

Why are beta-barrels commonly used for biotechnological applications? Give examples of such applications.

Answer 68

- they're stable - often topologically simpler than helices e.g., nanopore technology

Question 69

What can generally be said about the lipid composition in the outer leaflet compared to the inner leaflet of membranes?

Answer 69

Outer leaflet: cholesterol and glycolipids Inner leaflet: anionic lipids

Question 70

Why are lipids within membranes good drug targets?

Answer 70

Studying protein-lipid interactions can provide insights into how membrane proteins are inserted and anchored within the lipid bilayer, how they interact with other proteins and lipids, and how they carry out their functions. Such studies can also help us understand the effects of mutations or changes in the lipid composition of the membrane on protein function, and how these changes may contribute to various diseases. Furthermore, the understanding of protein-lipid interactions within membranes can aid in the development of new drugs that target membrane proteins. By understanding the interactions between specific proteins and lipids, we can design drugs that disrupt these interactions in specific locations e.g., targeting a specific GPCR within the brain as only in this location does it form interactions with lipid X.

Question 71

How might GPCRs be influenced by cholesterol? What is the evidence for this?

Answer 71

Membrane cholesterol has been shown to affect ligand binding, G-protein coupling, and intracellular signaling of GPCRs. The possible mechanism underlying the modulation of GPCR function by cholesterol could be via specific interaction of GPCRs with membrane cholesterol, or cholesterol-induced changes in global bilayer properties, or a combination of both mechanisms. Evidence for this is the large amount of XRC and cryoEM data that shows cholesterol bound to GPCRs, and the presence of CRAC motifs in many cholesterol-sensitive GPCRs (cholesterol recognition amino acid consensus).

Question 72

What is the role of PIP2 in bacterial Kir channels? What is the evidence for this?

Answer 72

A combination of CG and atomistic MD was used to model how PIP2 is able to activate Kir, yet inactivate other bacterial K channels. PIP2 interacts with the cytoplasmic domains of Kir channels, potentially aiding in stabilizing their open state.

Question 73

How might GPCRs be influenced by PIP2? What is the evidence for this?

Answer 73

A map was generated to see where PIP2 sits in a GPCR, allowing for mutations to be predicted that should prevent PIP2 from binding. This was confirmed using mass spec. Mass spec was also used to show that when a G protein bound this GPCR, more PIP2 molecules were pulled down. MD simulations were used to show that PIP2 binds the receptor, but also interacts with the G protein and this lowers the free energy of binding. The overall mechanism arose that PIP2 acts like a clamp to keep the G-protein bound to the GPCR and hence allow the signaling cascade to continue.

Question 74

How is PIP2 involved in RTK signaling?

Answer 74

When a ligand binds to an RTK, it causes the receptor to dimerize and become activated. This activation leads to the recruitment and activation of downstream signaling proteins, including PLCγ. PLCγ hydrolyzes PIP2 into two second messengers, (IP3) and diacylglycerol (DAG).

Question 75

What have simulations shown about how PH domains interact with the bilayer?

Answer 75

PH domains tend to interact with many lipids all at once, ensuring a solid interaction to begin the signaling cascade.

Question 76

What is PTEN?

Answer 76

A tumor suppressor protein that functions as a phosphatase. PTEN controls levels of PIP3 via dephosphorylation, decreasing the activity of downstream signaling. Loss of PTEN is frequently observed in many types of cancer, leading to uncontrolled cell growth and proliferation.

Question 77

What are secondary transporters?

Answer 77

Unlike primary active transporters that use ATP hydrolysis to directly move molecules across the membrane against their concentration gradient, secondary transporters use the pre-existing gradient of one molecule to transport another molecule against or along its own concentration gradient. e.g., symporters and anti-porters

Question 78

Describe the role of the Na+/glucose cotransporter. What's the stoichiometry? What type of transporter does the stoichiometry make this?

Answer 78

- uses the Na+ electrochemical gradient to drive uphill uptake of glucose - 2Na+: 1 glucose so electrogenic

Question 79

Give 3 examples of symporters.

Answer 79

1. Na+/K+/2Cl cotransporter 2. Na+/Cl- cotransporter 3. K+/Cl- cotransporter

Question 80

Give an example of an antiporter.

Answer 80

Na+/Ca exchanger

Question 81

What are the two main superfamilies of secondary transporters?

Answer 81

1. Major facilitator superfamily 2. Amino acid polyamine superfamily

Question 82

What is the consequence of inverted topology of membrane transporters?

Answer 82

The outward and inward open conformation of the transporters are energetically very similar. This enables transporters to shift their conformation easily between two extreme states.

Question 83

Describe the role of fucose permease.

Answer 83

A proton-coupled symporter

Question 84

Describe the role of LeuT.

Answer 84

A Na+-coupled amino acid symporter.

Question 85

Describe the role of VGLUT.

Answer 85

Vesicular glutamate transporters concentrate the excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential.

Question 86

How can allosteric binding of an ion or small molecule regulate a transporter?

Answer 86

1. Regulate access of ligands to the orthosteric site. 2. Alter the energetic barrier. NAMs and PAMs