Membranes

Question 1

Phospholipid Structure

Answer 1

• amphipathic molecules
• hydrophilic polar head
• head: choline, phosphate, glycerol
• hydrophobic non-polar fatty acid tails

Question 2

3 Phospholipids

Answer 2

1. phosphatidyl-ethanolamine
2. phosphatidyl-serine
3. sphingomyelin

Question 3

Sphingomyelin

Answer 3

• built from sphingosine
• fatty acid attached to amino acid group and phosphocholine attached to a terminal hydroxyl group
• free -OH can form H-bonds

Question 4

Sterol

Answer 4

• polar head group with rigid steroid ring structure and non polar hydrocarbon tail
• affects phospholipid compacting in membrane

Question 5

Glycolipids

Answer 5

• molecules modified via addition of sugars

eg. galactoserebroside and ganglioside

Question 6

Bilayer Formation

Answer 6

• polar molecules are hydrophilic and interact with water
• hydrophobic molecules force energetically unfavorable rearrangements of the water molecules
• energetic cost is reduced by hydrophobic molecule packing

Question 7

Membrane Properties

Answer 7

• 5-8 nm thick
• appear trilaminar
• fluid
• impermeable to large polar solutes + permeable to nonpolar small solutes

Question 8

Fluid Mosaic Model

Answer 8

Singer & Nicholson 1972
- individual lipid molecules are able to diffuse freely within bilayers
- researched using liposomes
liposomes = phospholipids in water form multilaminar vesicles with onion like bilayer arrangement

Question 9

Sonication

Answer 9

• applies high frequency sound energy and the structures in multilalamellar vesicles rearrange to make liposomes
• liposomes are stable, closed self-sealing solvent filled vesicles
• used to study membrane fluidity and individual movement of molecules in bilayer

Question 10

Photobleaching

Answer 10

• Fluorescence recovery after photobleaching (FRAP) in which fluorescent molecules or gold particles are attached to lipids (polar head groups)
• green fluorescent protein (gfp) emits green light when exposed to blue light that emits at 509 nm

Question 11

Lipid Movement

Answer 11

• lateral diffusion
• rotation
• flexion
• flip flop (not common)
• rapid lateral movement within one monolayer/leaflet
• diffusion coefficient of 10^-8 cm2/sec
• noncovalent interactions between lipid/protein molecules make movement rapid/easy

Question 12

Membrane fluidity + composition

Answer 12

• synthetic bilayers (one type of phospholipid) can have an induced change in physical condition into a 2D rigid crystalline gel state
• Phase Transition
• affected by tail length and double bond frequency

Question 13

Cholesterol

Answer 13

• interacts with regions of the fatty acid tails closest to the polar head group
• looser packing maintains fluidity at low temperature
• amount can be varied
• amount to which polar head goes into the tail region determines packing and therefore fluidity

Question 14

Lipid Domains

Answer 14

• certain lipid mixtures cause formation of transient domains
  eg. sphingomyelin, cholesterol

Question 15

Lipid Rafts

Answer 15

• cholesterol + sphingomyelin

- atomic force microscopy gives a contour map of the membrane to show these regions

Question 16

Membrane Asymmetry

Answer 16

• some lipids and proteins found predominantly in one leaflet
• asymmetry is not absolute
• important functional consequences
• apoptosis is triggered by movement of phosphotidylserine into the outer leaflet

Question 17

Membrane Proteins

Answer 17

• membrane proteins are asymmetric and this is absolute
• interact with membrane in many ways
• integral for biological function
• 50% of total membrane mass

Question 18

Transmembrane Proteins

Answer 18

• a helices compose the TM domain and exterior soluble regions
• single or multipass proteins
• can be beta barrels as well

Question 19

Peripheral Proteins

Answer 19

• monotopic, ie. only associate with one leaflet
• lipid modification acting as an anchor to one side
• form complexes with integral proteins

Question 20

a helices

Answer 20

• H bonds between residue N and residue N+4 (ie. bonds between carboxyl and amino groups) stabilise regular twisting structure
• maximises use of bond donors and acceptors
• all H bonds are intrahelical
• transmembrane a helical domains are hydrophobic
• specific amino acid residues: ile, leu, val, met
• 20 needed to make TM domain

Question 21

B barrels

Answer 21

• form from curved B strangs
• rigid structures acting as pores
• exclusively found in bacterial and mitochondrial membranes

Question 22

Protein Asymmetry

Answer 22

• specific orientation relative to membrane
• proteins only go one way up
  eg. B adrenogenic receptors binding epinephrine only function if ligand binding site is correctly oriented

Question 23

Detergent Solubilisation

Answer 23

• membrane proteins are difficult to study
• analysis requires soluble protein and membrane proteins are insoluble
• addition of a detergent creates membrane protein in bilayer and detergent micelles
• this creates a water soluble complex able to be isolated and studied

Question 24

Membrane Channels

Answer 24

• hydrophilic pores across the membrane
• narrow and highly selective
• 100 million ions/second

Question 25

Gating

Answer 25

1. voltage gated
2. ligand gated
3. mechanically gated

Question 26

Selectivity

Answer 26

• channel allows some ions/molecules to pass
• pores are narrow and often charged
• ions/molecules of appropriate size/charge can pass
• ions lose associated water molecules
• selectivity filter

Question 27

Channel Roles

Answer 27

1. fundamental to cell life
2. rapid responses
3. water transport

Question 28

Potassium Channels

Answer 28

• 4 monomers associated to form a homoquaternary protein
• each monomer has 2 TM regions and one half helix
• selective for K ions but not Na ions
• it is more favorable for the ion to pass to the selectivity filter and shed the hydration shell
• K ion is a perfect fit while the Na ion is too small for optimal interaction

Question 29

Mammalian K channel

Answer 29

• membrane potential sensed by voltage sensor domain containing positive amino acid residues
• pulled down to the negative cytosolic side of the membrane in a closed position
• if the outside is negative, the domain is pulled up and its connected loop is pulled down to open the pore

Question 30

Aquaporins

Answer 30

• water channels
• responsible for water secretion and reabsorption (kidney)
• flow rate = 10^9
• directionality determined by osmotic gradient
• 6 helices and 2.5 helices per monomer
• Positive charge of arginine prevent hydronium ion (+) from entering
• to prevent proton hopping and loss of membrane potential, 2 asparagine residues at the end of the half helices, each water forms transient interactions with both residues to prevent H+ addition

Question 31

Transporters

Answer 31

• carriers/permeases
• bind solute on one side of membrane, rearrange, and release the solute on the other side
• high affinity solute binding site

Question 32

Channels

Answer 32

• much weaker interaction with solute
• no directionality
• form aqeous pores across the membrane
• pores open and allow solute movement across membrane
• much faster transport

Question 33

Passive Transport

Answer 33

• facilitated diffusion

- electrochemical gradient needed for driving force

Question 34

Active Transport

Answer 34

• transport against gradient using pumps and ATP
• unidirectional solute movement
• coupling to source of metabolic energy
• coupled transporter - uses energy stored in ion gradient
• atp driven conformational change

Question 35

Ion Drive Pump

Answer 35

• uniport
  cotransport:
• symport or antiport

Question 36

Coupled Transporters

Answer 36

• harvest energy shared in the electrochemical gradient of one solute
• drives transport of other solute

Question 37

2 Types of Active Transport

Answer 37

1. Primary = driven by ATP hydrolysis
2. Secondary = couples downhill movement of solute with transport of another solute against its gradient
   - ion gradient has to be generated

Question 38

Lactose Permease

Answer 38

• 2 domains
• lactose and H= ions bind in cleft causing conformational change so molecule faces the other way
• ligand binding causes formation of a new salt bridge that forces opening a different way

Question 39

Enzyme Coupled Receptors

Answer 39

• ligand binding leads to dimerization of inactive receptor in activate a catalytic domain
• ion channel coupled receptors
• G protein coupled receptors

Question 40

GPCR

Answer 40

• helical bundle with specific ligand binding site
• high affinity interactions
• induced conformational change activates intracellular G protein
• a, B, y subunits with a,y anchored to membrane

Question 41

G Protein Activation

Answer 41

• inactive form, a binds GDP
• ligand binding catalyses GDP - GTP exchange
• a subunit conformational change so it no longer has high affinity for the complex and dissociates

Question 42

Downstream Signalling

Answer 42

• complex binds to adenylate cyclase that catalyses activation of cyclic AMP
• cyclic AMP activates protein kinase A which goes on to affect many cellular processes including protein expression

Question 43

G Protein Reset

Answer 43

• GTP is hydrolysed and complex dissociates from the adenyl cyclase to reform a trimer
• receptor ligand dissociates
• active form is phosphorylated leads to B-arrestin binding to physically block further GCPR binding