Membrane ALL Flashcards
Overview/importance
- 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
Environment + topologies
- 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)
Tm helix insertion
- Hydrophobic core = 30A thick, 20aa
- Ribosome, translocon (TM segments shunted sideways, gate btw TM2+7, plug = TM2A)
- Hydrophobic residues can be inserted
- Prediction
Principle of membrane structure
- Analyse known structure of membrane to derive statistical rules
- Idea of length
Key residues at interface
- Trp/tyr in porin + ion channels, can form H bond
- lys ‘snorkelling’
Lipids
- 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
Expression
- Need ↑ amounts
- Over-expression can be tricky
- Multiple sequence alignment to look for bacterial homologue
Detergents
- 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
Crystallisation
- Lipid cubic phase (curved 3D liquid crystalline structure that self-assembles, stabilises proteins)
- Monoolein = often used
Alternatives to crystallisation
- 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
X ray diffraction
- Hard to crystallise
- Detergents means have weak lactic forces so ↓ ordered, ↓ resolution
- Nanobody/lipid cubic phase
Cryo-EM
- Single particle EM, freeze + look at structure, 3D info
- Statistical sorting
- 3.3A resolution
- Shorter time
Solution NMR
- Small membrane proteins that x crystallise + too small for cryo-EM
- Solubilise w/ detergent, different structure
MD
- Simulate flexibility of protein at room temperature
- Can look at interactions in cell-like environment
Biological roles of ion channels
- Axons have Na+ + K+ that are switched on/off
- Action potential activates Ca2+ channels → release Ca2+ → neurotransmitter fusion
Key properties of channel
- TM protein that forms pore
- Some selectivity
- Filter that interacts w/ favoured ions only
- Switch btw open + closed w/ gate or ligand
K+ channel topology
- 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
Selectivity
- 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
Mechanism for K+ passing through the channel
- K+-H20-K+ as if K+ occupied S1-4 would be unstable
2. All sites occupied w/ K+ + instability means ions move quickly
Voltage gating
- 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
Overview
- Channel + pore = small once Δ during transport
- Transporter = TM conf change
- Pump = catalytic events drive Δ
Aquaporins
- 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)
Water conduction
- 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
Transporter
- 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
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
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
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
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
Elevator-mechanism transporter
- Scaffold domain + transport domain
- Open state (gate open, occluded state up, gate moves + solute leaves
- Alternating access mechanism
Cys loop family receptor summary
- Shared topology
- Found mostly in neuromuscular synapses and brain
- Associated w/ ↑ disease
- Excitatory/cation selective or inhibitory/anion selective