Membrane proteins

Question 1

membrane function

Answer 1

protect cell from toxicity
let specific things into cell
separate incompatible processes

Question 2

Integral membrane protein

Answer 2

Span whole membrane

secondary structure

Question 3

Peripheral membrane protein

Answer 3

span only half of protein

hydrophobic tail anchor

Question 4

Phospholipid

Answer 4

glycerol backbone
fatty acids
X group - provide diversity (hydrophobic, signalling)

Question 5

Fluid mosaic model

Answer 5

fast lateral diffusion

Question 6

Leaflet

Answer 6

inner or outer surfaces -> varies with organism

POP, ptd seriine

Question 7

Types of integral proteins

Answer 7

1 - N terminal out, C terminal in
2 - C terminal out, N terminal in
3 - multipass

Question 8

hydropathy plot

Answer 8

hydropathy score vs amino acid score
rolling average over short window
-> in membrane, predict helices

Question 9

Membrane protein expression challenges

Answer 9

appropriate expression system (right lipids=right host)
make work in aqueous solution
flexible and dynamic

Question 10

detergents

Answer 10

lipids with tails -> micelle

determine best using SEC - not aggregated, not fragmented

Question 11

lipid nanodisks

Answer 11

mimic native environment

belt protein + phospholipid surface

Question 12

Activation of G proteins

Answer 12

GTP->GDP

alpha and beta/gamma subunits dissociate

Question 13

alpha subunit

Answer 13

PLC - phospholipase C -> PIP2

• > DAG (hydrophobic) -> PKC = phosphorylation
• > IP3 -> ER Ca2+

Question 14

PKC domains

Answer 14

pleckstrin homology - +ve pocket = active site
C2 - binds next to Ca2+, ptd serine = bound to membrane
C1 - Zn2+ ions, binds DAG

Question 15

non-specific protein-lipid interactions

Answer 15

Addition of lipids (or head groups) = membrane association

stable of dynamic

Question 16

Specific protein-lipid interactions

Answer 16

Specific binding domains - signalling
ions for stability/regulation
recognise head groups for targeting

Question 17

protein signalling

Answer 17

oligomerisation or conformational change
optimised for regulation not throughput
second messengers -> cellular behaviour

Question 18

oligerimisation

Answer 18

receptor tyrosine kinase = dimerisation -> activation

Question 19

conformational changes

Answer 19

type 3 proteins
off and on states
GPCRs

Question 20

heterotrimeric g protein complex

Answer 20

alpha beta and gamma

alpha + gamma = ptm ->lipids

Question 21

GPCRs

Answer 21

7 transmembrane helices
intracellular C, extra N
glycosylated and disulphide bonds
intracellular loops = binding site G protein

Question 22

Classes GPCR

Answer 22

A = Rhodopsin family =small molecule ligands -> orthosteric site
B = Secretin family - polypeptide hormones/proteins
C = Glutamate family = orthosteric binding site (additional domain)
F = frizzled

Question 23

how to get crystal structure of GPCR

Answer 23

trap in one position = high affinity agonist, remove GPP, fusion protein, nano body from llama

Question 24

GPCR alpha binding site

Answer 24

TM 6 and 3 move apart, 5 and 7 together

very subtle

Question 25

solving structure

Answer 25

crystal structure or cryo electron-microscopy (images in buffer, depends on resolution), x-ray crystallography

Question 26

arrestin signalling

Answer 26

GRK5 activated -> receptor phosphorylated -> arrestin recruitment -> desensitised -> endocytosis -> dephosphorylation/recycling/degradation

Question 27

rhodopsin

Answer 27

eye responds to light - cis -> trans = active

Question 28

partial agonists

Answer 28

different equilibrium R -> R*1 -> R*2 eg B2 | unique active state R -> R*1 or R -> R*2 eg A2a

Question 29

biased ligands

Answer 29

drive towards B-arrestins or G protein pathway | use to remove adverse effects eg opioids and analgesics

Question 30

passive transport

Answer 30

down concentration gradient simple - gases, small polar/hydrophobic facilitated- channel or transporter (large polar charged)

Question 31

active transport

Answer 31

against concentration gradient primary - pumps secondary - cotransport

Question 32

vesicular transport

Answer 32

large quantities, less selective exocytosis endocytosis

Question 33

Channels

Answer 33

can be gated or open to both sides, governed by concentration gradient

Question 34

transporters

Answer 34

``` passive or active alternating access (lock) ```

Question 35

uniporter

Answer 35

[low] binds -> [high] via energy from ATC hydrolysis or photons change in conformation

Question 36

symporter

Answer 36

[low] -> [high] coupled with [high] -> [low] | harness other electrochemical gradients

Question 37

antiporter

Answer 37

[low] -> [high] coupled with [high]

Question 38

MFS

Answer 38

major facilitator superfamily alternating access transporters 4 x 3 transmembrane helices -> two domains polar cavity

Question 39

glucose transporters

Answer 39

GLUT1-4 use proteoliposomes to characterise, wash and quantity contents after transport (measure competition) C-terminal domain = bend and clamp N-terminal domain = rocking

Question 40

signal transduction

Answer 40

cell signalling ions (different conc and speed) polarisation = K+/Na+ pumps and channels ATPases

Question 41

K+ channel

Answer 41

selective for K+ over Na+ (bigger + Na+ stronger bond to water) square anti prism C=O binding domain x 4 (glycine for flexibility) stabilised by helix dipole 2 at a time + pushed through by repulsion

Question 42

types of ion channels

Answer 42

voltage gated, ligand gated, mechanical stimuli (deformities, touch, sound, osmotic pressure)

Question 43

X-ray crystallography vs cyro-electron microscopy

Answer 43

both need secondary purified protein X-ray needs crystal, cyro needs liquid cyro needs big protein (50 kDa) electron density map vs images