membrane proteins

Question 1

What do Sterols do to the membrane?

Answer 1

Cholesterol found in humans acts to reduce felxibilty of the billayer and become more ridged.

Question 2

What are sphingolipids?

Answer 2

major components of neuronal membranes and are found primarily in plasma membranes. cause differnt phase behavior in membranes including the formation of local domains.

Question 3

What are glycolipids?

Answer 3

found exclusively on the outside of cells and are synthesised in the ER and golgi.

Question 4

Why do lipids form bilayers and what forms if its only a single layer?

Answer 4

bilayered due to the hydrophobic and hydrophillic nature of the tails and head. (head is hydrophillic)

a single layer forms a micelle

Question 5

What percentage of the human genome codes for membrane proteins?

Answer 5

26-36%

Question 6

How thick is the bilayer and how many amino residues are needed to cross the membrane?

Answer 6

the bilayer is 36A thick and each amino acid is around 1.5A so need 24aa to cross the bilayer

Question 7

How long are helical domains typically? and what effects their orientations in the bilayer?

Answer 7

Typically 19-24aa in length. the transmembrane domains tend to be hydrophobic and their aromatic resiudes tend to be at the surface. The orrientation of the domain tends to depend on the length of the transmembrane domain and the thickness of the bilayer.

Question 8

What makes a beta barrel?

Answer 8

comprised of anti parallel b-sheets rolled into a barrel.
beta sheets are extended so only need 7 residues to cross the bilayer.
typical strands are 9-11 residues long and are slanted at 45 degrees.
is a stable structure and the size of the pore is determined by number of strands

Question 9

what are the two types of loops found on beta barrels and whats characteristic about them?

Answer 9

there are extracellular loops that tend to be long and have high sequence variability

there are periplasmic loops that are typically smaller and formed by tight loops and turns.

Question 10

What are the two classes of porins

Answer 10

1) general diffusion, not very selective, determined by size of pore, rate of transfer linked to conc gradient
2) Substrate specific, selective, exhibit michaelis menten kinetics - typically larger than 18 strands. tend to be voltage gated.

Question 11

What is an example of a non specific baterial porin and its characterisitcs?

Answer 11

OmpF.
General diffusion – limited by molecular weight of substrate (e.g. OmpF), rate of transport determined by concentration gradient

Typically 105 copies per cell

Expression determined by osmolarity

115 kDa, forms a homo-trimer

16 antiparallel strands, tilted by 45 degrees

Loop 3 fold back into channel and has a highly conserved PEFGG motif

Loop constricts pore to 15x22A

Cluster of acidic residues complemented by a basic motif on the beta-barrel to from a transverse electric gradient

Question 12

What is an example of a substrate specific bacterial porin?

Answer 12

LamB/Maltroporin

18 stranded porin

Classical structure{short periplasmic loops and long extracellular loops

Outer face of beta-barrel, covered with largely uncharged groups (grey – carbon)

Pore constricted by 3 loops, confiring an hour glass shape to the pore

Question 13

cerebroside is a glycolipid and these are found where?

Answer 13

exclusively found on the outside of cells. they are synthesised in the ER/Golgi

Question 14

what is a Ganglioside?

Answer 14

A ganglioside is a molecule composed of a glycosphingolipid (ceramide and oligosaccharide) with one or more sialic acids (e.g. N-acetylneuraminic acid, NANA) linked on the sugar chain.

basically more complex branched oligosacharides

Question 15

what analysis techneique was used to determine the bilayer structure of membranes?

Answer 15

X ray/Neutron diffraction.

Question 16

What is the overall role of membrane proteins such as ligand gated channels, tyrosine kinase receptors and G protein coupled receptors?

Answer 16

transfer of information as they are involved in cell signalling.

Question 17

What are some examples of helical membrane proteins?

Answer 17

bateriorhodopsin, rhodopsin, H+/Cl- channels, His Kinase receptors, aquaporins, transporters (LacY), channels (KcsA), Atpases.

Question 18

What is the length of an alpha helical transmembrane domain?a

Answer 18

each amino acid gives a rise of 1.5A. the bilayer is approx 36A. takes at least 24 residues to cross the bilayer.

Question 19

What is the distribution of residues in a helical transmembrane protein?

Answer 19

hydrophobic residues tend to be located within the hydrophobic core of the membrane with the large aromatic residues at the water/bilayer interface.

Question 20

What degrees does helical packing tend to take place?

Answer 20

between +5 and +25 degrees.

Question 21

What are the typical crossing angles found in helical packing?

Answer 21

-37, +83, and +22 degrees

Question 22

What configuration of packing is typically preffered?

Answer 22

ANTIPARRALEL.

Question 23

What are the promotion sequences for helix interactions of small sidechains and polar residues?

Answer 23

SMALL side chains: GXXXG

POLAR residues: SxxSSxxS (serine is a small ammino acid with a hydroxyl group so can form hydrogen bonds.

Question 24

What does shear number of a beta barrel mean?

Answer 24

the change of residue numbers on a Beta strand when a point moves in the left hydrogen bond direction back to the same beta strand.

think of it like the next strand is longer/ shorter to stop the barrel from buldging.

Question 25

What is the typical tilt found in a beta barrel?

Answer 25

45degrees.

Question 26

describe the structure of a beta barrel?

Answer 26

a beta barrel is made up of typically an even number of beta strands ordered in a antiparralel config with a 45 degrees tilt. they have short periplasmic connections and long external loops.

the surface of a beta barrel is aliphatic side chains that form a hydrophobic ring. the barrel has high sequence variability and the external loops are mobile with high sequence variability.

Question 27

What are the two classes of porins?

Answer 27

1) general diffusion. not very selective, is determined by the size of the pore. rate of transfer is linked to the concentration gradient.
2) Substrate specific, selective, exhibit Michaelis menten kinetics. they tend to be typically larger than 18 strands. tend to be voltage gated.

Question 28

Give an example of a non specific bacterial porin.

Answer 28

OmpF.
typically 10^5 copies per cell.
is 115kDa and is a homo-trimer.
16 antiparallel strands tilted by 45 degrees.
loop 3 fold back into channel and has a highly conserved PEFGG motif.
loops constricts size to 15x22A

Question 29

What is an example of a substrate specific bacterial porin.

Answer 29

LamB/Maltoporin.
18 stranded porin with classical structure (short periplasmic long extracellular etc)
outer face of barrel is covered with largely uncharged groups.
pore is constricted by 3 loops confiring an hour glass shape to the pore.

Question 30

How does Lamb/Maltoporin transport subtrate down its barrel?

Answer 30

via greasy slide. formed by 6 aromatic residues on side of pore.

framed by 2 ionic tracks of acidic and basic residues.

Question 31

What is an ionic track?

Answer 31

ionic tracks have 3 major sugar binding sites.
aromatics interact with the hydrophobic surfaces of the sugars and hydrogen bonding stabalises the hydroxyl groups on the sugar.
oligosaccharides can twist through the pore.

the sugars move along a network of H bonds. (think like one gets formed them moved and then formed again kinda like how actin moves)

Question 32

What beta barrel is an EXCEPTION to the classical structure and function.

Answer 32

VDAC a mitocondrial beta barrel protein. they do not contain lipiopolysaccarides and have very little variation between loops in VDACs. N terminus extensions are more common.

Question 33

What are some of the differences between VDAC and bacterial porins

Answer 33

VDAC contains 19 transmembrane strands (odd) which infers that some of the strands are also in a parallel arrangment.

they are voltage gated at small transmembrane voltages

have 1NMR and 2Crystal strucutres.

flexible N terminus (small alpha helix making close contact with barrel wall.)

they are coded for by nucular DNA.

Question 34

How are VDACs selective?

Answer 34

they have pore lined with positive charge accounting for ATP/ADP selectivity

Question 35

how long do alpha helical membrane protiens tend to be?

Answer 35

19 to 24 residues in length.

Question 36

Where are the aromatic residues found in an alpha helical membrane protein?

Answer 36

at the bilayer surface

Question 37

WHat determines the oientation of alpha helical membrane proteins?

Answer 37

length of transmembrane domain and bilayer. thickness.

Question 38

WHat is KcsA and describe its strucutre

Answer 38

A prokaryotic potassium channel.
tetramer strucutre with 3 helical domains and 2 transmembrane domains.

inverted teepee structure. is constricted at the cytoplasmic face and open at the extracellular face.

Question 39

What is a motif?

Answer 39

a shot conserved sequence pattern associated with distinct functions of a protein or DNA.

Question 40

What is a selectivity filter in ion selectivity?

Answer 40

a conduction pathway formed from a conserved TVGYG motif. allows the backbone carbonyl groups to point towards the channel lining/potassium ions.

Question 41

how was KcsA the potassium channel first isolated and purified?

Answer 41

it was expressed in bacteria and solubilized in decylmaltoside. then got tagged with His to aid purification and then the C terminus was removed, gel filtered and exchanged into LDAO. (Lauryldimethylamine oxide)

Question 42

where can potassium channels be found?

Answer 42

neurons,
cardiac tissues,
epithelial cells

Question 43

what are the regulatory domains of KcsA?

Answer 43

N terminus and C terminus

Question 44

what domains do KcsA have?

Answer 44

3 helical domains

2 transmembrane domains

Question 45

what side of the KcsA protein channel is constricted

Answer 45

the cytoplasmic side

Question 46

how is the pore of the KcsA channel formed?

Answer 46

there are shorter half helices at the top of the channel that make a pore. their negative C terminus are pointed towards the inside of the channel that creates a dipole that allows stabalisation of the ions that would normally be energetically unfavourable in the middle of the channel

Question 47

What Motif allows for ion selectivity?

Answer 47

TVGYG. means the carbonyl groups point towards the channel lining.

Question 48

what do the carbonyl groups do in the potassium channel?

Answer 48

ligate K+ by mimicing the water molecules alowing the conduction of the ions based on fluctuations of the groups since the backbone is quite ridgid.

Question 49

Why can Na+ not pass potassium channels

Answer 49

they are smaller ions so cannot be effectively ligated by the Carbonyl groups.

Question 50

Why are the ions not conducted adjacently in the channel?

Answer 50

if the ions were conducted adjacently they would cause elctrostatic repulsion. they are only conducted in alternate states.

Question 51

the potassium channel MthK has what domains that regulate the gating?

Answer 51

RCK which is a calcium ion sensing domain. has 4 copies per monomer and 2 attached to the channel. makes a gating ring of 8 RCK domains.