Membranes Extra

Question 1

Types of fold

A helix

Answer 1

• 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

Question 2

Types of fold

B barrel

Answer 2

• 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

Question 3

Types of fold

Other

Answer 3

• Some have both

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

Question 4

Membrane protein vs water soluble protein structure

Answer 4

• 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

Question 5

Topology + structure prediction

Answer 5

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

Question 6

Issue w/ B-barrel

Answer 6

• 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

Question 7

Issues w/ structure prediction

Answer 7

• 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

Question 8

Potassium channel structure extra

Answer 8

• 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

Question 9

Selectivity potassium channel extra

Answer 9

• 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

Question 10

Gating

Answer 10

• 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

Question 11

Sodium structure channel

Answer 11

• 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

Question 12

Sodium selectivity

Answer 12

• 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

Question 13

Sodium gating

Answer 13

• 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

Question 14

Calcium channel structure

Answer 14

• Similar to sodium

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

Question 15

Calcium selectivity

Answer 15

• EEEE sequence near sodium channel EDEKA motif

Question 16

Calcium gating

Answer 16

• S4 also controls

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

Question 17

Calcium inactivation = different

Answer 17

• 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