Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MBB266 > Baker (Membrane proteins) > Flashcards

Baker (Membrane proteins) Flashcards

1
Q

What biological processes do membrane proteins participate in?

A
  • 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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How are major classes of membrane proteins identified?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the topology of many membrane proteins?

A
  • N-ter is extracellular domain
  • 2-3 AAs traverse membrane and form membrane spanning α helices
  • C-ter is cytosolic domain
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What does it mean to say AAs have diff hydopathies?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the Kyte and Doolittle scale?

A
  • from Arg = -4.5 to Ile = +4.5

- value of at least 1.6 for TM helix

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How can hydropathy plots predict membrane spanning α-helices?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Why is putting β- sheets into membrane unfavourable, and how is this resolved?

A
  • NH/C=O not H bonded at each edge

- forms β-barrel

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the pattern of hydrophobic/hydrophilic residues in β-barrels?

A
  • inside hydrophobic by having alt pattern of hydrophobic and hydrophilic residues
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Why can’t hydropathy plots predict β-barrels?

A
  • half hydrophobic and half hydrophilic
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are lipid anchors?

A
  • post translational mods

- hydrophobic anchors enable some proteins to covalently link to lipid bilayer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are the diff types of lipid anchors, and where are they found?

A
  • acylation = inner leaflet of euks
  • prenylation = inner leaflet of euks
  • thioester = inner leaflet of euks
  • GPI anchor = exterior leaflet of euks
  • bacterial lipoproteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are acylation lipid anchors?

A
  • 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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are prenylation lipid anchors?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are thioester lipid anchors?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are GPI anchor lipid anchors?

A
  • mod C-ter w/ ethanolamine linked to oligosaccharide linked to inositol of phosphatidyl inositol
  • exoplasmic face
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are bacterial lipoprotein lipid anchors?

A
  • thioester and amide linkage
  • +vely charged n region
  • hydrophobic h region
  • lipobox w/ invariant Cys C-region
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

How permeable is the membrane to diff types of molecules?

A
  • 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are the 2 types of membrane transport proteins?

A
  • ATP powered pumps

- ion channels (can be gated)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are the diff types of transporters?

A
  • 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
20
Q

What is osmosis?

A
  • flow of water from compartment w/ low solute conc to 1 w/ high solute conc
21
Q

Why are aquaporins needed?

A
  • 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
22
Q

What is the structure of aquaporins and how was this discovered?

A
  • X-ray crystallography and e- crystallography
  • 6 TM helices and 2 short helices per subunit
  • tetramer of 4 subunits
  • each subunit has pore
  • DIAGRAM*
23
Q

How do aquaporins have specificity for water?

A
  • 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)
24
Q

Why is it important that aquaporins have specificity for water?

A
  • don’t want to transport eg. H3O+ as would mess up ion conc across membrane
25
Q

Why are proteins not cotranslated?

A
  • 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*
26
Q

What is the electrochemical grad in cells in diff permeability situations?

A

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

27
Q

How do transport proteins function together in mammalian cells?

A
  • Na+/K+ pump –> driven by ATP hydrolysis
  • K+ channel –> sets up ec grad
  • Na+/lysine symporter –> pumps Lys uphill using Na+ ec grad
28
Q

What is the structure of K+ channel in Streptomyces lividans?

A
  • 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*
29
Q

How does K+ selectivity filter work?

A
  • 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
30
Q

How are K+ ions found in the K+ channel?

A
  • 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
31
Q

How do membranes facilitate compartmentalisation of biomolecules and pathways?

A
  • topology v important
  • if 1 membrane then inside same as outside of cell, eg. golgi
  • if 2 membranes, inside same as cytosol, eg. mito
32
Q

What are the types of membrane protein, there characteristics, and an example?

A
  • 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
33
Q

Does orientation of protein change between synthesis in RER and in final destination?

A
  • no
34
Q

How do single pass type I membrane proteins work?

A
  • ribosome attaches to pore on membrane
  • signal peptidase assoc w/ membrane
  • keeps posting mRNA through until reach stop-transfer anchor seq
35
Q

How do single pass type II membrane proteins work?

A
  • 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
36
Q

What are some seq analysis methods for membrane proteins?

A
  • 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
37
Q

What is the signal seq?

A
  • short peptide present at N-ter of majority of newly synthesised proteins (not long enough to go across membrane)
38
Q

What are some experimental techniques for determining membrane protein topology?

A
  • enzyme tags –> reporter proteins
  • chemical mod
  • antibodies against protein epitopes
  • microscopy (EM)
  • N or C-ter tags w/ GFP
  • protease accessibility
39
Q

What are the diff reporter proteins?

A
  • β-galactosidase (LacZ)
  • E. Coli alkaline phosphatase (PhoA)
  • β-lactamase
  • N-glycosylation scanning
40
Q

How can β-galactosidase be used as a reporter gene?

A
  • 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
41
Q

How can E. Coli alkaline phosphatase be used as a reporter gene?

A
  • 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
42
Q

How can β-lactamase be used as a reporter gene?

A
  • 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
43
Q

How can N-glycosylation scanning be used as a reporter gene?

A
  • 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
44
Q

How does cysteine mutagenesis scanning work?

A
  • 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
45
Q

How can cysteine mutagenesis scanning detect a periplasmic Cys residue, and what is the disadvantage?

A
  • Cys specific membrane permeable label
  • Cys specific membrane impermeable block
  • disadv = need controls, eg. inside out vesicles, detergent
46
Q

How can cysteine mutagenesis scanning detect a cytoplasmic Cys residue, and what are the advantages?

A
  • Cys specific membrane permeable label
  • Cys specific membrane impermeable block
  • adv = active protein, in vivo studies poss, small changes to protein
47
Q

What are other methods of determining protein topology?

A
  • 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

MBB266 (7 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions