Membrane ALL Flashcards
(77 cards)
1
Q
Overview/importance
A
- 20% genes, 50% drug targets
- <1% structures determined
- Many = a-helical or bacteria outer-membrane proteins like OmpA = B-sheet
- 25-30% of all genes
- Diseases
2
Q
Environment + topologies
A
- Single TM can oligomerise
- Membrane associated proteins
- Integral membrane proteins
- Environment is important
- 2/3 state folding model (2o structure means peptide can satisfy backbone H bond requirements)
3
Q
Tm helix insertion
A
- Hydrophobic core = 30A thick, 20aa
- Ribosome, translocon (TM segments shunted sideways, gate btw TM2+7, plug = TM2A)
- Hydrophobic residues can be inserted
- Prediction
4
Q
Principle of membrane structure
A
- Analyse known structure of membrane to derive statistical rules
- Idea of length
5
Q
Key residues at interface
A
- Trp/tyr in porin + ion channels, can form H bond
- lys ‘snorkelling’
6
Q
Lipids
A
- Phosphatidylcholine to complex like PIP2
- Varies btw membranes
- Need good match btw hydrophobic protein + surrounding lips
- 1st shell of lipid = restricted
- Bacteriorhodopsin has hole occupied by up to 6 lipids
7
Q
Expression
A
- Need ↑ amounts
- Over-expression can be tricky
- Multiple sequence alignment to look for bacterial homologue
8
Q
Detergents
A
- Keeps membrane protein in soluble form
- Needs to be sufficiently disruptive to remove phospholipid but x Δ conf of protein
- Most have hydrophilic head (makes water soluble) + non-polar tail, bile acid has both polar + non-polar ‘faces’
- octyl glucoside = useful, DDM = good
- Micellisation
9
Q
Crystallisation
A
- Lipid cubic phase (curved 3D liquid crystalline structure that self-assembles, stabilises proteins)
- Monoolein = often used
10
Q
Alternatives to crystallisation
A
- Amphipols = polymers w/ hydrophobic + hydrophilic regions
- Nanodisc = used in cryo-EM , scaffold protein forms 2 belts that make stable environment, incorporate protein inside
- nanobody = add H20-soluble protein
11
Q
X ray diffraction
A
- Hard to crystallise
- Detergents means have weak lactic forces so ↓ ordered, ↓ resolution
- Nanobody/lipid cubic phase
12
Q
Cryo-EM
A
- Single particle EM, freeze + look at structure, 3D info
- Statistical sorting
- 3.3A resolution
- Shorter time
13
Q
Solution NMR
A
- Small membrane proteins that x crystallise + too small for cryo-EM
- Solubilise w/ detergent, different structure
14
Q
MD
A
- Simulate flexibility of protein at room temperature
- Can look at interactions in cell-like environment
15
Q
Biological roles of ion channels
A
- Axons have Na+ + K+ that are switched on/off
- Action potential activates Ca2+ channels → release Ca2+ → neurotransmitter fusion
16
Q
Key properties of channel
A
- TM protein that forms pore
- Some selectivity
- Filter that interacts w/ favoured ions only
- Switch btw open + closed w/ gate or ligand
17
Q
K+ channel topology
A
- Conserved core topology
- Central pore-forming region - M1, loop that goes in + out of membrane, M2
- 4 subunits come together, M2 form lining of core pore
- TVGYG
18
Q
Selectivity
A
- M2 close off channel + positions
- All carbonyl O point the same way
- As K+ enters, encounters 8O (S4), 8O(S3), 8O(S2), 8O(S1) and 4O(So)
- In solution, K+ surrounded by 8H20
- Replaced by 8O in protein = selective = no E lost
- Na+ is smaller so H20 have stronger interaction, x fit as well, more expensive to dehydrate
19
Q
Mechanism for K+ passing through the channel
A
- K+-H20-K+ as if K+ occupied S1-4 would be unstable
2. All sites occupied w/ K+ + instability means ions move quickly
20
Q
Voltage gating
A
- TVGYG in filter
- Conserved Glycogen in inner helix → bend/hing in middle of helix, opens channel
- S4 has repeat of R (+ve charge)
- S1-4 move when change voltage
- Pulls S4 helix down when membrane changes voltage, pulls on S4/5 linker → opens channel
21
Q
Overview
A
- Channel + pore = small once Δ during transport
- Transporter = TM conf change
- Pump = catalytic events drive Δ
22
Q
Aquaporins
A
- Water selective, high H20 permeability
- Structure = tetramer, each subunit has 6TM connected w/ 5 loops
- 3 helices where 3rd = re-entrant, then 6,5,4 where 6 = re-entrant too
- Loop B + E have conserved NPA motif needed to maintain proton gradient
- ar/R site = selectivity filter
- NPA orient water
- x allow protons through, main barrier = NPA, 2nd = ar/R
- Large solutes excluded
- Experiment
- Glpf = glycerol selective (↑ glycerol permeability, ↓ H20 than Aqp)
23
Q
Water conduction
A
- hAqp4 1.8A structure
- 2 1/2 helices w/ NPA motif
- Breaks H bond chain of H20 in centre, prevents H+ conduction against column of water
- Can adopt alternative conformation + break H bonded chain
- Glpf = also tetramer, central constriction pore
- Polar region of pore interacts w/ OH of glycerol, hydrophobic interacts w/ hydrophobic
24
Q
Transporter
A
- Uniporter, symporter or antiporter
- P type ATPase (gradient of key cation like Na/K+,
- Conserved DKTGTLT + TGES motives
- 10 TM helices, region in cytoplasmic side responsible for ATPase catalytic machinery
- Ca2a+ ATPase
- ATP bound to catalytic site opens TM region to Ca2+ from inside cell
25
Overview
- Evolution of alternating access model = transport protein consists of entity within membrane that either faces outward + binds substrate on outer face of membrane or undergoes conf change
26
ATP binding cassette transporter
- 2 Tm region for transport, 2 NBD that hydrolyses ATP → free E to drive conf change
- 2 NBD engage in symmetric dimer w/ 2 ATP molecules sandwiched in the dimer
- e.g. type I ABC importers responsible for nutrient uptake, ATP hydrolysis drives conf change
- Export mechanism (inward facing, bind ATP, flips NBD away → Δ access of bs from inward to outward, ADP dissoc
- Import (closed → occluded, conformational Δ that moves accessibility of B-12 bound site to inward facing, catalytic region open to ATP, another Δ that releases B-12 → closed ATP free → outward open
27
Secondary transporter
| 1. MFS
- Includes uni- sym- and antiporters
- Transport small solutes
- Lactose permeate = common structure of 12TM helices btw 2 domains
- Sugar switches to inward conf
- 2 domains rock back and forward
- Mechanism (FucP, proton binds Asp, sugar binds outward open → proton jumps + triggers conf change, sugar disc → reverse back to open)
- Alternating access mechanism = 2 major conf inward + outward facing, NTD + CTD change position relatively
28
Secondary transporter
| 2. LeuT superfamily
- Sodium symporters
- Internal symmetry, 2x5TM + surrounding helices that x form part of mechanism
- bs for Na+ + solute = at interface btw 2 domains
- OUTWARD = water penetrates from outside, gate shut inside
- INWARD = opposite
- OCCLUDED = pockets can be occupied by ions/H20 but closed on both sides
- DAT
29
Elevator-mechanism transporter
- Scaffold domain + transport domain
- Open state (gate open, occluded state up, gate moves + solute leaves
- Alternating access mechanism
30
Cys loop family receptor summary
- Shared topology
- Found mostly in neuromuscular synapses and brain
- Associated w/ ↑ disease
- Excitatory/cation selective or inhibitory/anion selective
31
Structure of AchR
- 5 subunits w/ 3 distinct regions (EC, TM + IC)
- Muscle have 2a,y,gamma + B, Neuronal = mostly 3B +2a
- EC = 4TM per subunit, IC loop btw M3 + M4, M4 faces lipid, M2 lines pore
- Acetyl choline binds btw a+b, a+y interface
- Ach bs = ABC + EDF, different segments
- Cys loop = btw 2 lys
- Mutation of M2 helices
32
ELIC/GLIC
- 3.4A GLIC
- Similar to nACHr
- GLIC opened at low pH
- ELIC has more occlusion by Phe or Leu
GluCI = glutamate-gated chloride channel, anion selective, ambiguous conformation
SHT3 = crystallised w/ Ab
GABA B3 = closed, B3 x physiological
GlyR = solved w/ cryo-EM, has 2 gates, when channel opens, radius is large enough to allow ion through
33
How to know closed state
- nAChr has hydrophobic section near 9' region
- Narrow point, r=3.1A, appears open
- Narrow pore means water x pass
- As ↑ radius, ↑ chance of fully open channel
- Ir could add dipoles to pore-lining surface
- Opening = overlay ELIC + GLIC, quaternary twist
34
Why bacterial channels x help
- ECD of ELIC suggests AchBP = basal state, most have agonist bound
- Conf of TMS of nACHR EM is closer to GLIC than ELIC → active state?
- ELIC = unusual
35
Lipids influence nACHR
- As ↑ cholesterol, ↑ stabilisation in resting state
| - If no anionic lipids, nachR stabilised in desensitised state
36
Receptor responses can be 'tuned'
- Different sequences of receptor affect response to agonist in different ways
- Achieved w/ alternative splicing
- Editing = in critical position
37
Disease
- SCS, atrophy of muscle, prolonged channel activation
38
Ionotropic glutamate receptors
- Ligand-gated ion channels
| - Functions in brain, when things go wrong → disease
39
Classification
- uglu, slower responses
- 4 main families = alpha, kainate, NMDA, orphan
- In vivo, Glu opens for all
40
iGluR structure
- Tetrameric w/ 4-fold symmetry
- Extracellular portion = dimer of dimers
- Ligand binding domain
- Homologue to KcsA
Mechanism (in closed state shut, Glu binds cleft triggers channel to open, D2 close, M3 move into open state)
41
Structural data
- Physiological + x-ray structures
- MD simulations (App state)
- Different flexibility to different agonists
- Many structures solved as dimers w/ Gly+Thr linker rather than whole Tm
42
Open state
- CT2 blocks desensitisation, trap in open/closed
| - btw EC + TM region = ↑ dynamic, hard to resolve
43
Overall motion
- Glu receptor solved in open + closed, 10A
- Resting → open = contraction of receptor, twisting motion
- Dimer interface = important
- Block w/ cyclothiazide
- Use in therapy
44
RNA editing
- Alternative splicing at 'flip flop' site
- Affects desensitisation
- Q/R site
- R/G editing
45
NMDA receptor
- Only active under certain conditions
- High permeability to Ca2+
- 2 agonists to open
- Receptor = hetramer
- GluNI can make functional channels
- NMDA receptors have at least 6 regulatory sites for ligands
- Glutamate more effect than NMDA
- Important in learning
- Agonist used in Alzheimers
- Signal to noise hypothesis
46
Signalling overview
- Signal binds 7TM, GPCR binds G protein + activates → a+B subunits
- 800 GPCRs
- ↑ range of responses
- Important drug target
47
Classification
1. homology (Classes, classA = largest, rhodopsin-like, classC = metabotropic glutamate)
2. Graf system
3. Genetic structure (1st no = what helix residue of interest is on)
48
Rhodopsin
- Found in rod cells
- 2 glycosylation sites at NTD, N-2/15
- 2 palmitoylation sites at C
- Lys 296
- Glycosylation on inside disc
- Conversion of light (light causes retinal to isomerase, lys → trans, 1 rhodopsin activates 100 transducers, CGMP hydrolysed, allows Na+ to open,
49
Dynamics
- Dynamic so ↑ conformational heterogeneity, hard to crystallise
- Basal activity at low concentration of drug
50
Non-rhodopsin structure
- B2 -adrenergic receptor
| - Removed long flexible loop 3, lipid cubic phase
51
Key differences btw different states
- Ionic lock (DRY/ERY at iC of TM3, salt bridge to E6/40 in inactive state)
- Breaks upon activation
- NPxxY on TM4
- PIF motif
52
Activation
- Lock opens, outward movement of TM5+6
- Mutant E113Q, TM5/6 move away
- Resting → intermediate state
- Ionic lock x always broken
- NPxxY conserved
- Pif has subtle movement
53
Arrestin
- Ligand induced conf change in GPCR facilitates interaction w/ G protein
- GPCR = GEF for Ga
- Second messengers = activated by effectors
- B-arresting binds to phosph GPCR
- Cells become desensitised
- Agonist can bind and activate state that would be phosphorylated → arresting bound
54
Lipid composition
- Cholesterol + anionic lipids = important
- PIP2 has phospholipid, inositol groups + 2 P
- Lipids vary in head groups + FA tails
55
Lipid ion channel structure
- Anionic lipids influence function of KV channel
- Cryo- EM of GABA ↑ res → PIP2 bound
- ANT1 binds CDL
- Free E landscape and see how Δ w/ different lipids
- Kir
- Pip2 mechanism
56
Cholesterol
- Many GPCRs bind cholesterol
57
PIP2
- -vely charged lipids could aid in allosteric activation of GPCR receptors
- GPCR add to MD w/ PIP2, show where PIP2 likely to bind
- conserved bs in class A receptors
- GPCR bound w/ PIP2
58
Signalling
- EGFR → EGF binds → 7M helix dimer → tyrosine kinase domai
| - TK autophosph → Ras → P13K → PIP3→PIP2 → ds signalling
59
PTEN protein
- 2 domains, phosphatase _ C2
- Both bind PIP
- Tumour suppressor
60
Receptor tyrosine kinase
- TM = reconstitution experiment
- Glycolipids like GM3 inhibit EGFR activation, PIP2 promotes dimerisation + activation
- Mutations in basic region weaken int. w/ GM3
Stimulate juxtamembrane in bilayer
61
Types of fold
| A helix
- A-helix bundle protein = 20-25% of genes of most organisms
- H bonds form btw N-H group of aa and C=O of aa 4 residues earlier, satisfied requirement
- 20 residue stretch
- Antiparallel association
- 3o and 4o structure
62
Types of fold
| B barrel
- Largely found in OM of gram -ve bacteria + mitochondria
- Antiparallel B sheets, H bonds satisfied
- Even no.
- Alternating polar + hydrophilic aa so favourable
- E.g. porin = 16-18 B-strands
- Structurally harder to keep
- Longer loops on outside
63
Types of fold
| Other
- Some have both
| - E.g. new fold in bacteria = combination of 2a-helical + B-barrel folds → a-helical barrel
64
Membrane protein vs water soluble protein structure
- Membrane proteins = hydrophobic + relatively insoluble
- Water soluble also fold into bundles of a-helices similar to membrane
- But, outer section of water have hydrophilic aa but hydrophobic aa are buried (opposite in membrane)
- Membrane proteins hard to purify
- Some hydrophobic aa have similar structure to hydrophilic ones
- Keep interior residues the same so overall structure maintained
- Could cause subtle changes = drawback
65
Topology + structure prediction
- Hard to get 3D structure (crystallisation, native environment)
- Hydropathy plot: taken window of 20aa and calculate mean hydrophobicity, shift across 1 and repeat
66
Issue w/ B-barrel
- B-strands are shorter and less conspicuous than a-helices
- Also ↑ structural variants, barrels = 8-36 strands
- Other B-sheet rich regions like pre-barrel region
- Use neural networks: training set of proteins answer Y/N proteins
67
Issues w/ structure prediction
- Hard to discriminate btw TM helices + other hydrophobic features
- TMH in single-spanning proteins = ↑ hydrophobic than polytopic membrane, can disrupt topology if x taken into account
- Some structures too complicated to fit into simple models e.g. 310 helix
68
Potassium channel structure extra
- Common feature = pore forming domain + regulatory domain
- Tetramer w/ 4 single domain that have 2 helices (M1+2) w/ short loop, central pore that runs down centre of channel of M2
- Pore region has selectivity filter, water-filled cavity + closed gate
- Selectivity filter = TVGYG, O of which point into centre (S1-4)
- S1-4 form VSD
- S5-6 = like M1/2 = pore forming domain
- S4 = +ve Arg, connected to S4/5 linker
- Glycine wings
- Lipids btw S1-4 voltage domain
69
Selectivity potassium channel extra
- The potassium ion radius = 1.33A, sodium = 0.95A, size not enough to discriminate
- Thought to do with dipoles (The magnitude of the repulsive interaction btw 2 ligands coordinating an ion is sensitive to the electrostatic properties of the ligands)
- Ligand-ligand repulsion
70
Gating
- IC gate includes helix-bundle crossing, EC = selectivity filter
- Resting = both gates closed
- +ve S4 helix pulled down to attract -ve charge in cell
- Membrane depolarised → S4 moves up → transient bridges formed → 310 conformation→ S6 interacts w/ linker
Closing
- S4 moves inward, ions move out of pore → hydrophobic collapse → S4/5 moves fully down
71
Sodium structure channel
- Less known
- Single polypeptide chain folds into 4 homologous repeats (each of 6TM repeats)
- Can have other subunits like B
- DEKA selectivity in eukaryotes, EEEE in prokaryotes
- Bacteria + human = only 25% sequence homology, x know structure
72
Sodium selectivity
- Domains contribute asymmetrically (III and IV contribute more than I and II)
- Selectivity depends on field strength of binding site, high field strength ion like Glu needed to ↑ Na+ selectivity
73
Sodium gating
- Unkown
- Also due to TM movement changes due to S4 → S4/5 linker opening IC gate
- Prokaryotes also interactions w/ CTD
- Eukaryotic sodium channels have short IC loop connecting S6 of III to S1 of IV = inactivation gate
74
Calcium channel structure
- Similar to sodium
| - a subunit = also 4 domains each of 6 TM helices
75
Calcium selectivity
- EEEE sequence near sodium channel EDEKA motif
76
Calcium gating
- S4 also controls
| - Also forms globular domain by CTF + III-IV linker
77
Calcium inactivation = different
- Both voltage-gated + calcium gated
- After prolonged depolarisation → conformational change → inactivation shield, Ca2+ x enter
- When Ca enters, Ca domain formed near start of pore, when fully loaded w/ Ca, calmodulin interacts w/ sites of NTD → inactivated
