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Paper 1 > Membrane ALL > Flashcards

Membrane ALL Flashcards

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
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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)
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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
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4
Q

Principle of membrane structure

A
  • Analyse known structure of membrane to derive statistical rules
  • Idea of length
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5
Q

Key residues at interface

A
  • Trp/tyr in porin + ion channels, can form H bond

- lys ‘snorkelling’

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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
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7
Q

Expression

A
  • Need ↑ amounts
  • Over-expression can be tricky
  • Multiple sequence alignment to look for bacterial homologue
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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
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9
Q

Crystallisation

A
  • Lipid cubic phase (curved 3D liquid crystalline structure that self-assembles, stabilises proteins)
  • Monoolein = often used
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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
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11
Q

X ray diffraction

A
  • Hard to crystallise
  • Detergents means have weak lactic forces so ↓ ordered, ↓ resolution
  • Nanobody/lipid cubic phase
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12
Q

Cryo-EM

A
  • Single particle EM, freeze + look at structure, 3D info
  • Statistical sorting
  • 3.3A resolution
  • Shorter time
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13
Q

Solution NMR

A
  • Small membrane proteins that x crystallise + too small for cryo-EM
  • Solubilise w/ detergent, different structure
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14
Q

MD

A
  • Simulate flexibility of protein at room temperature

- Can look at interactions in cell-like environment

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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

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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
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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
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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
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19
Q

Mechanism for K+ passing through the channel

A
  1. K+-H20-K+ as if K+ occupied S1-4 would be unstable

2. All sites occupied w/ K+ + instability means ions move quickly

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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
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21
Q

Overview

A
  • Channel + pore = small once Δ during transport
  • Transporter = TM conf change
  • Pump = catalytic events drive Δ
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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)
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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
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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
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25
Q

Overview

A
  • 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
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26
Q

ATP binding cassette transporter

A
  • 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
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27
Q

Secondary transporter

1. MFS

A
  • 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
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28
Q

Secondary transporter

2. LeuT superfamily

A
  • 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
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29
Q

Elevator-mechanism transporter

A
  • Scaffold domain + transport domain
  • Open state (gate open, occluded state up, gate moves + solute leaves
  • Alternating access mechanism
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30
Q

Cys loop family receptor summary

A
  • Shared topology
  • Found mostly in neuromuscular synapses and brain
  • Associated w/ ↑ disease
  • Excitatory/cation selective or inhibitory/anion selective
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31
Q

Structure of AchR

A
  • 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
Q

ELIC/GLIC

A
  • 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
Q

How to know closed state

A
  • 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
Q

Why bacterial channels x help

A
  • 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
Q

Lipids influence nACHR

A
  • As ↑ cholesterol, ↑ stabilisation in resting state

- If no anionic lipids, nachR stabilised in desensitised state

36
Q

Receptor responses can be ‘tuned’

A
  • Different sequences of receptor affect response to agonist in different ways
  • Achieved w/ alternative splicing
  • Editing = in critical position
37
Q

Disease

A
  • SCS, atrophy of muscle, prolonged channel activation
38
Q

Ionotropic glutamate receptors

A
  • Ligand-gated ion channels

- Functions in brain, when things go wrong → disease

39
Q

Classification

A
  • uglu, slower responses
  • 4 main families = alpha, kainate, NMDA, orphan
  • In vivo, Glu opens for all
40
Q

iGluR structure

A
  • 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
Q

Structural data

A
  • 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
Q

Open state

A
  • CT2 blocks desensitisation, trap in open/closed

- btw EC + TM region = ↑ dynamic, hard to resolve

43
Q

Overall motion

A
  • Glu receptor solved in open + closed, 10A
  • Resting → open = contraction of receptor, twisting motion
  • Dimer interface = important
  • Block w/ cyclothiazide
  • Use in therapy
44
Q

RNA editing

A
  • Alternative splicing at ‘flip flop’ site
  • Affects desensitisation
  • Q/R site
  • R/G editing
45
Q

NMDA receptor

A
  • 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
Q

Signalling overview

A
  • Signal binds 7TM, GPCR binds G protein + activates → a+B subunits
  • 800 GPCRs
  • ↑ range of responses
  • Important drug target
47
Q

Classification

A
  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
Q

Rhodopsin

A
  • 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
Q

Dynamics

A
  • Dynamic so ↑ conformational heterogeneity, hard to crystallise
  • Basal activity at low concentration of drug
50
Q

Non-rhodopsin structure

A
  • B2 -adrenergic receptor

- Removed long flexible loop 3, lipid cubic phase

51
Q

Key differences btw different states

A
  • 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
Q

Activation

A
  • 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
Q

Arrestin

A
  • 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
Q

Lipid composition

A
  • Cholesterol + anionic lipids = important
  • PIP2 has phospholipid, inositol groups + 2 P
  • Lipids vary in head groups + FA tails
55
Q

Lipid ion channel structure

A
  • 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
Q

Cholesterol

A
  • Many GPCRs bind cholesterol
57
Q

PIP2

A
  • -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
Q

Signalling

A
  • EGFR → EGF binds → 7M helix dimer → tyrosine kinase domai

- TK autophosph → Ras → P13K → PIP3→PIP2 → ds signalling

59
Q

PTEN protein

A
  • 2 domains, phosphatase _ C2
  • Both bind PIP
  • Tumour suppressor
60
Q

Receptor tyrosine kinase

A
  • 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
Q

Types of fold

A helix

A
  • 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
Q

Types of fold

B barrel

A
  • 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
Q

Types of fold

Other

A
  • Some have both

- E.g. new fold in bacteria = combination of 2a-helical + B-barrel folds → a-helical barrel

64
Q

Membrane protein vs water soluble protein structure

A
  • 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
Q

Topology + structure prediction

A
  • Hard to get 3D structure (crystallisation, native environment)
  • Hydropathy plot: taken window of 20aa and calculate mean hydrophobicity, shift across 1 and repeat
66
Q

Issue w/ B-barrel

A
  • 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
Q

Issues w/ structure prediction

A
  • 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
Q

Potassium channel structure extra

A
  • 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
Q

Selectivity potassium channel extra

A
  • 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
Q

Gating

A
  • 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
Q

Sodium structure channel

A
  • 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
Q

Sodium selectivity

A
  • 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
Q

Sodium gating

A
  • 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
Q

Calcium channel structure

A
  • Similar to sodium

- a subunit = also 4 domains each of 6 TM helices

75
Q

Calcium selectivity

A
  • EEEE sequence near sodium channel EDEKA motif
76
Q

Calcium gating

A
  • S4 also controls

- Also forms globular domain by CTF + III-IV linker

77
Q

Calcium inactivation = different

A
  • 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

Paper 1 (12 decks)

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