Membrane proteins Flashcards
membrane function
protect cell from toxicity
let specific things into cell
separate incompatible processes
Integral membrane protein
Span whole membrane
secondary structure
Peripheral membrane protein
span only half of protein
hydrophobic tail anchor
Phospholipid
glycerol backbone
fatty acids
X group - provide diversity (hydrophobic, signalling)
Fluid mosaic model
fast lateral diffusion
Leaflet
inner or outer surfaces -> varies with organism
POP, ptd seriine
Types of integral proteins
1 - N terminal out, C terminal in
2 - C terminal out, N terminal in
3 - multipass
hydropathy plot
hydropathy score vs amino acid score
rolling average over short window
-> in membrane, predict helices
Membrane protein expression challenges
appropriate expression system (right lipids=right host)
make work in aqueous solution
flexible and dynamic
detergents
lipids with tails -> micelle
determine best using SEC - not aggregated, not fragmented
lipid nanodisks
mimic native environment
belt protein + phospholipid surface
Activation of G proteins
GTP->GDP
alpha and beta/gamma subunits dissociate
alpha subunit
PLC - phospholipase C -> PIP2
- > DAG (hydrophobic) -> PKC = phosphorylation
- > IP3 -> ER Ca2+
PKC domains
pleckstrin homology - +ve pocket = active site
C2 - binds next to Ca2+, ptd serine = bound to membrane
C1 - Zn2+ ions, binds DAG
non-specific protein-lipid interactions
Addition of lipids (or head groups) = membrane association
stable of dynamic
Specific protein-lipid interactions
Specific binding domains - signalling
ions for stability/regulation
recognise head groups for targeting
protein signalling
oligomerisation or conformational change
optimised for regulation not throughput
second messengers -> cellular behaviour
oligerimisation
receptor tyrosine kinase = dimerisation -> activation
conformational changes
type 3 proteins
off and on states
GPCRs
heterotrimeric g protein complex
alpha beta and gamma
alpha + gamma = ptm ->lipids
GPCRs
7 transmembrane helices
intracellular C, extra N
glycosylated and disulphide bonds
intracellular loops = binding site G protein
Classes GPCR
A = Rhodopsin family =small molecule ligands -> orthosteric site B = Secretin family - polypeptide hormones/proteins C = Glutamate family = orthosteric binding site (additional domain) F = frizzled
how to get crystal structure of GPCR
trap in one position = high affinity agonist, remove GPP, fusion protein, nano body from llama
GPCR alpha binding site
TM 6 and 3 move apart, 5 and 7 together
very subtle
solving structure
crystal structure or cryo electron-microscopy (images in buffer, depends on resolution), x-ray crystallography
arrestin signalling
GRK5 activated -> receptor phosphorylated -> arrestin recruitment -> desensitised -> endocytosis -> dephosphorylation/recycling/degradation
rhodopsin
eye responds to light - cis -> trans = active
partial agonists
different equilibrium R -> R1 -> R2 eg B2
unique active state R -> R1 or R -> R2 eg A2a
biased ligands
drive towards B-arrestins or G protein pathway
use to remove adverse effects eg opioids and analgesics
passive transport
down concentration gradient
simple - gases, small polar/hydrophobic
facilitated- channel or transporter (large polar charged)
active transport
against concentration gradient
primary - pumps
secondary - cotransport
vesicular transport
large quantities, less selective
exocytosis
endocytosis
Channels
can be gated or open to both sides, governed by concentration gradient
transporters
passive or active alternating access (lock)
uniporter
[low] binds -> [high]
via energy from ATC hydrolysis or photons
change in conformation
symporter
[low] -> [high] coupled with [high] -> [low]
harness other electrochemical gradients
antiporter
[low] -> [high] coupled with [high]
MFS
major facilitator superfamily
alternating access transporters
4 x 3 transmembrane helices -> two domains
polar cavity
glucose transporters
GLUT1-4
use proteoliposomes to characterise, wash and quantity contents after transport (measure competition)
C-terminal domain = bend and clamp
N-terminal domain = rocking
signal transduction
cell signalling
ions (different conc and speed)
polarisation = K+/Na+ pumps and channels ATPases
K+ channel
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
types of ion channels
voltage gated, ligand gated, mechanical stimuli (deformities, touch, sound, osmotic pressure)
X-ray crystallography vs cyro-electron microscopy
both need secondary purified protein
X-ray needs crystal, cyro needs liquid
cyro needs big protein (50 kDa)
electron density map vs images
