Baker (Membrane proteins) Flashcards
What biological processes do membrane proteins participate in?
- ion pumps and channels –> reg ionic balance of cell
- carriers across membrane
- cell surface receptors –> recognition of extracellular hormones and signalling systems
- conveyors of cell identity –> in immunological reactions
- converters of energy stimuli
How are major classes of membrane proteins identified?
- take membrane fraction by changing salt conc/pH and seeing which proteins dissociate
- increase salt concs to decrease IMFs between peripheral and integral proteins –> identifies peripheral
What is the topology of many membrane proteins?
- N-ter is extracellular domain
- 2-3 AAs traverse membrane and form membrane spanning α helices
- C-ter is cytosolic domain
What does it mean to say AAs have diff hydopathies?
- free energies for transfer of AA in α-helix from membrane interior to water
- largest value most hydrophobic
- smallest (most -ve) values are charged AAs
What is the Kyte and Doolittle scale?
- from Arg = -4.5 to Ile = +4.5
- value of at least 1.6 for TM helix
How can hydropathy plots predict membrane spanning α-helices?
- window of 19 residues used to calc hydropathy of that stretch of polypeptide
- usually 19 as this is no. req to get across membrane
- lipid membrane diff so can req more/less
- when looking at plots not always perfect hydrophobicity, as if protein has function , eg. transport, might need certain residues that aren’t hydrophobic, eg. to H bond
- if helices diagonal need more AAs to get across membrane
Why is putting β- sheets into membrane unfavourable, and how is this resolved?
- NH/C=O not H bonded at each edge
- forms β-barrel
What is the pattern of hydrophobic/hydrophilic residues in β-barrels?
- inside hydrophobic by having alt pattern of hydrophobic and hydrophilic residues
Why can’t hydropathy plots predict β-barrels?
- half hydrophobic and half hydrophilic
What are lipid anchors?
- post translational mods
- hydrophobic anchors enable some proteins to covalently link to lipid bilayer
What are the diff types of lipid anchors, and where are they found?
- acylation = inner leaflet of euks
- prenylation = inner leaflet of euks
- thioester = inner leaflet of euks
- GPI anchor = exterior leaflet of euks
- bacterial lipoproteins
What are acylation lipid anchors?
- myristoylation = amide bond to N-ter Gly
- myristoyl acid (C14 CA) embedded in bilayer
- normally co-translational (after N-ter Met removed)
- can also occur irreversibly w/ palmitic acid (C16)
What are prenylation lipid anchors?
- thioester link to C-ter Cys
- polymers of isoprene linked to Cys near C-ter
- C-ter seq is -Cys-a-a-X-
- aaX cleaved after attachment
What are thioester lipid anchors?
- thioester link to Cys easy to cleave
- reversible (by thioesterases)
- occur sw/ C14, C16, C18 CAs
- no consensus seq, but often occurs close to acylation/prenylation sites
What are GPI anchor lipid anchors?
- mod C-ter w/ ethanolamine linked to oligosaccharide linked to inositol of phosphatidyl inositol
- exoplasmic face
What are bacterial lipoprotein lipid anchors?
- thioester and amide linkage
- +vely charged n region
- hydrophobic h region
- lipobox w/ invariant Cys C-region
How permeable is the membrane to diff types of molecules?
- gases (eg. CO2) = permeable
- small uncharged polar molecules (eg. ethanol/water) = permeable
- large uncharged polar molecules (eg. glucose) = slightly permeable
- ions (eg. K+) = impermeable
- charged polar molecules (eg. AAs/ATP/NAs/proteins) = impermeable
What are the 2 types of membrane transport proteins?
- ATP powered pumps
- ion channels (can be gated)
What are the diff types of transporters?
- uniporter = 1 down conc grad
- symporter = 1 down and 1 against conc grad, both same direction
- antiporter = 1 down and 1 against conc grad, diff directions
What is osmosis?
- flow of water from compartment w/ low solute conc to 1 w/ high solute conc
Why are aquaporins needed?
- cells semipermeable to water w/o water transporters in membrane
- so if no aquaporins and cell in hypotonic solution (low solute conc) cell lysis as water can’t leave
What is the structure of aquaporins and how was this discovered?
- X-ray crystallography and e- crystallography
- 6 TM helices and 2 short helices per subunit
- tetramer of 4 subunits
- each subunit has pore
- DIAGRAM*
How do aquaporins have specificity for water?
- pore diameter 2.8Å, = VDW diameter of water, H3O+ wouldn’t fit
- highly conserved Arg195 and His180 form gate and attract water
- pore lined w/ hydrophobic residue to facilitate transport (so no interactions)
Why is it important that aquaporins have specificity for water?
- don’t want to transport eg. H3O+ as would mess up ion conc across membrane
Why are proteins not cotranslated?
- if water channel in aquaporins contains uninterrupted chain of water molecules then H+ also transported by “proton conducting wire”
- causes transfer of charge to inside of cell (=BAD)
- 2 crucial conserved Asn residue H bond donors to water in channel, breaking wire
- waters have to take particular orientation so can’t bind to each other
- DIAGRAM*
What is the electrochemical grad in cells in diff permeability situations?
Membrane impermeable to Na+/K+/Cl-:
- membrane electrical pot = 0mV
- high K+ in cytosol and high Na+ in extracellular medium
Membrane permeable only to Na+ (Na+ channels):
- membrane electrical pot = +59mV (cytosolic +ve)
- Na+ flows down conc grad to cytosol
- charge separation across membrane
Membrane permeable only to K+ (K+ channels):
- membrane electrical pot = -59mV
- K+ flows down conc grad to extracellular medium
- imbalance of ions gives electrical grad as well as chem grad = ec pot
In resting cells pm lots K+ channels and v few Na+ and Cl- channels
How do transport proteins function together in mammalian cells?
- Na+/K+ pump –> driven by ATP hydrolysis
- K+ channel –> sets up ec grad
- Na+/lysine symporter –> pumps Lys uphill using Na+ ec grad
What is the structure of K+ channel in Streptomyces lividans?
- 4 identical subunits each w/ 2 membrane spanning helices
- 1 channel per tetramer
- P segment selectivity filter
- -> homologous in all K+ channels
- -> mutations stop selectivity
- -> functional protein if bacterial P segment replaced w/ mammalian P segment
- up to 10^8 ions per sec transport rates
- 10Å diameter vestibule in tetrameric channel and 3Å diameter selectivity filter
- DIAGRAM*
How does K+ selectivity filter work?
- not just based on size, as Na+ smaller than K+
- K+ transported as desolvated ion
- energy cost in desolvating matched by specific coord to main chain CO groups from P segment lining channel
- Na+ too small to allow perfect coord w/ channel CO groups, so desolvation energy cost would be too high
- diff in activation energy favous K+ transport over Na= by factor greater than 1000
How are K+ ions found in the K+ channel?
- 4 binding sites for K+
- alt occupation of sites 1 and 3 or 2 and 4
- little energy diff between K+ in diff sites
- electrostatic repulsion of K+ drives throughput of ions
- large vestibule on intracellular side minimises distance K+ have to travel through hydrophobic membrane
How do membranes facilitate compartmentalisation of biomolecules and pathways?
- topology v important
- if 1 membrane then inside same as outside of cell, eg. golgi
- if 2 membranes, inside same as cytosol, eg. mito
What are the types of membrane protein, there characteristics, and an example?
- I = long end in exoplasmic space, eg. insulin receptor
- II = long end in exoplasmic space, eg. transferrin receptor
- III = long end in cytosol, COO- in cytosol and long COO- end, eg. cytochrome P450
- I, II, III all single pass, usually α helix, NH3 or COO- can be on outside of cell
- IV = no leader seq to direct protein to go outside, eg. GPCRs
- GPI linked protein = multipass, NH3+ and COO- could be either way round
Does orientation of protein change between synthesis in RER and in final destination?
- no
How do single pass type I membrane proteins work?
- ribosome attaches to pore on membrane
- signal peptidase assoc w/ membrane
- keeps posting mRNA through until reach stop-transfer anchor seq
How do single pass type II membrane proteins work?
- protein never goes through translocon as no signal seq
- bounces round in cytosol until get same hydrophobic residue that form TM α helix and then recognised by translocon
- +ve AAs interact w/ -ve side of membrane
What are some seq analysis methods for membrane proteins?
- hydropathy plots to try and predict membrane spanning helices
- look for +ve AAs either side of helix in seq (“+ve inside rule”)
- predict N-ter signal seq
- find topology by finding which way some TM seqs are then can work out topology of rest of membrane
What is the signal seq?
- short peptide present at N-ter of majority of newly synthesised proteins (not long enough to go across membrane)
What are some experimental techniques for determining membrane protein topology?
- enzyme tags –> reporter proteins
- chemical mod
- antibodies against protein epitopes
- microscopy (EM)
- N or C-ter tags w/ GFP
- protease accessibility
What are the diff reporter proteins?
- β-galactosidase (LacZ)
- E. Coli alkaline phosphatase (PhoA)
- β-lactamase
- N-glycosylation scanning
How can β-galactosidase be used as a reporter gene?
- cytoplasmic protein –> 4 subunits, active as tetramer, correct folding and active when in cyto
- fuse LacZ to C-ter truncated membrane protein of interest, following activity w/ X-Gal
- if LacZ in cyto get blue stain
How can E. Coli alkaline phosphatase be used as a reporter gene?
- used for prok or euk proteins expressed in proks
- periplasmic protein w/ N-ter leader seq –> 2 subunits active as dimers, 2 disulphides per subunit, incorrect folding and inactive in cyto
- fuse PhoA to C-ter truncated membrane protein of interest
- follow activity w/ X-P
How can β-lactamase be used as a reporter gene?
- periplasmic protein
- monomeric
- protects cells against action of β-lactam antibiotics
- fuse β-lactamase to C-ter truncated membrane protein of interest
- follow activity w/ cell growth assays on ampicillin plates
- reqs complementary fusions and controls
How can N-glycosylation scanning be used as a reporter gene?
- euk proteins N-glycosylated in ER by oligosaccharide transferase
- adds sugars to amino group of Asn in NxT/S seq on luminal group of membrane protein
- delete pot glycosylation sites by SDM assay for glycosylation by SDS/PAGE electrophoresis or mass spec
- can also be used in C-ter deletion fusion proteins
- Asn must be 12 residues upstream or 14 downstream of membrane for OST to function
How does cysteine mutagenesis scanning work?
- introd Cys mutants to membrane protein
- probe mutant protein w/ membrane permeable and membrane impermeable reagents
- native Cys residues must be mutated to Ser
- mutant protein usually active
- can use reagents linked to biotin, fluorescent molecules or radiolabels
How can cysteine mutagenesis scanning detect a periplasmic Cys residue, and what is the disadvantage?
- Cys specific membrane permeable label
- Cys specific membrane impermeable block
- disadv = need controls, eg. inside out vesicles, detergent
How can cysteine mutagenesis scanning detect a cytoplasmic Cys residue, and what are the advantages?
- Cys specific membrane permeable label
- Cys specific membrane impermeable block
- adv = active protein, in vivo studies poss, small changes to protein
What are other methods of determining protein topology?
- use antibodies raised to natural or engineered epitopes on membrane protein
- immunoaffinity chromatography = labels on exoplasmic face of protein will bind to column
- tag GFP to protein and probe w/ fluorescence microscopy
- use EM to directly visualise protein = label protein w/ antibody then use gold particles coated w/ protein A, which binds non-specifically to Fc region
