8: Membrane proteins

Question 1

What are cells enclosed by?

Answer 1

Lipid bilayer membranes

Question 2

Bacterial cell difference

Answer 2

lacking internal membrane enclosed organelles (nuceleus, mitochondria)

Question 3

What type of bacteria have an inner and outer membrane?

Answer 3

gramm-negative bacteria

Question 4

What distinguishes Gram-positive bacteria from Gram-negative bacteria?

Answer 4

Gram-positive have no outer membrane

Question 5

What is the thickness of the lipid bilayer?

composition?

Answer 5

Approximately 33 ±3 Å

> charged groups on outside, hydrophobic inner regions

Question 6

How do lipid compositions vary among cellular membranes?

Answer 6

They are distinct in different membranes

Question 7

What are the two types of membrane proteins?

Answer 7

Peripheral Membrane Proteins (at surface on inside side)
> Membrane associated
> Membrane anchored

Integral Membrane Proteins (a-helix/b-barrel spans through whole lipid bilayer)

Question 8

What percentage of the membrane area is occupied by proteins?

Answer 8

15-35%

Lipid:protein ratio varies

Question 9

how is the plasma membrane structured?

How do lipid molecules behave in the plasma membrane?

Answer 9

into domains
> membrane behaves as compartmentalised fluid

Molecule movement:
short term diffusion within compartment, long term hop diffusion between compartments

Question 10

How does translational diffusion in the cell membrane compare to liposomes?

Answer 10

It is slower in the cell membrane

Question 11

What contributes to the organization/structure of the plasma membrane?

Answer 11

Membrane skeleton proteins (e.g., actin, spectrin)
Anchored proteins
Induced oligomerization

= formation fo domains

Question 12

Peripheral membrane proteins

Answer 12

=

Question 13

What mediates the interaction of associated peripheral membrane proteins?

Answer 13

Amphiphilic helices (+/- end on both side) or charged residues

Question 14

Tail anchored peripheral membrane proteins

about them? eg?

Answer 14

Covalently attached to bilayer via anchor
> embedded in membrane
> 0.5% of human proteins = palmitoylated
> e.g. ras proteins

Question 15

integral membrane proteins

Answer 15

=

Question 16

integral membrane proteins
%?
two fundamental types?

Answer 16

20-30% of all proteins

Beta-barrels (outer membrane) and alpha-helical bundles (inner membrane)

Question 17

Integral membrane proteins environment (vs cytosol)

5 facts
idk

Answer 17

Cytosol vs Plasma membrane
> Isotropy only in cytosol (same property in all directions)
= pH and redox gradient in membrane
> more proteins in membrane than cytosol
> dielectric constant in cytosol = 80 = H2O
> membrane has no partners for salt, h, disulfide bridges

Question 18

What is harder to do in the membrane environment?

Answer 18

break main-chain hydrogen bonds
[ionise salt chain
break a salt bridge]

Question 19

what is easier to do in the membrane environment?
and why (2)

Answer 19

expose hydrophobic group
bring subunits in close proximity
= Favour formation of hydrogen bonded secondary structure !!

due to low dielectric constant and nonpolar domains

Question 20

Alpha helical membrane proteins
topology

Answer 20

Bitopic = span across membrane once
> Typ1: N = Outside
> Typ2: N = inside
Polytopic = protein spans multiple times across = Typ3

Typ4: several polypeptides/proteins each with 1 transmembrane helix
= Oligomeric

Question 21

Transmembrane helix key property?

Answer 21

Hydrophobic
> long stretches of hydrophobic = possible transmembrane helix

Question 22

Alpha helical membrane protein properties

3

Answer 22

Distribution of charged and aromatic residues
> core of hydrophobic surrounded by aromatic/charged (outside bilayer)

Number of TM helix varies
> conserved within family
> 12 = preferred for transporters

Helix-Helix interactions
> mostly hydrophobic
> hydrophobic effect NOT stabilising (unlike sol. proteins)
> guided by surface complementarity/vDW

Question 23

What role do glycine and proline play in transmembrane helices?

Answer 23

They allow close packing in helices

Question 24

Proline in protein-alpha-helix and transmembrane alpha-helix

Answer 24

Proline
= rare in soluble protein alpha helix
= common in TM alpha helix
> centre of bilayer
> creates 120 degree kink

Question 25

What are the properties of transmembrane helices? shorter

Answer 25

Mostly hydrophobic Association guided by surface complementarity Few hydrophilic residues present

Question 26

What motif allows close helical packing in transmembrane helical bundles?

Answer 26

GXXG/A motif > Helix-Helix IA

Question 27

What types of repeats do helical membrane proteins often contain?

Answer 27

Inverted repeats and parallel repeats

Question 28

Beta-barrel

Answer 28

=

Question 29

What are the characteristics of β-barrel membrane proteins? aka Outer MP = OMP 3

Answer 29

8-22 strands (even) Antiparallel strands High sequence variability = harder to predict than alpha m.p

Question 30

What are the characteristics of transmembrane strands in β-barrel proteins? 3

Answer 30

Alternating hydrophobic and hydrophilic residues Hydrophobic outside Hydrophilic inside (alpha = hydrophobic transmembrane region)

Question 31

What are the interactions between membrane proteins and lipids? example

Answer 31

Charged/Aromatic residues mostly outside bilayer > surround hydrophobic core (TM) > interactions are specific and functionally relevant e.g. aquaporin = water channel

Question 32

Membrane protein insertion: alpha helix 5 steps

Answer 32

1. Ribosome undergoing synthesis (starts from N) 2. signal recognition particle SRP recognises N-terminal hydrophobic sequence of Alpha-helix + pauses Translation 3. SRP directs ribosome to SR (SRP-Receptor) in cytoplasm 4. Ribosome is transferred to the Sec61 translocon and resumes translation 5. Sec forms lateral gate to membrane for alpha-helix > Helix orientation upon insertion!! > Translation continues during and after membrane insertion, enabling multiple transmembrane domains to be inserted sequentially.

Question 33

What is the role of the Sec translocon (2) ? and how?

Answer 33

It inserts alpha-helical membrane proteins > accepts nascent proteins (still being synthesised) > allows for different topologies of alpha-helix, as they're only orientated during insertion

Question 34

What happens upon signal helix binding in the Sec translocon?


Answer 34

Conformational changes occur in the translocon > SecYEG forms a lateral gate to membrane for alpha-helix

Question 35

How are outer membrane proteins inserted? 4 steps

Answer 35

1. OMP synthesised in cytosol 2. Unfolded OMPs pushed through SecY: req ATP > Chaperones help transport unfolded OMP by keeping it in unfolded state 3. hand over unfolded OMP to BAM complex (beta barrel assembly machine) in outer membrane 4. unfolded OMP associates with BAMa and is folded here until independent barrel is formed > association through C-terminal Beta recognition signal > barrel formed with BAM help, once it is stable enough it leaves without need of additional proteins Unfolded OMP → SecYEG → chaperones → BAM complex → C-terminal β-signal → BAM-mediated β-barrel insertion

Question 36

What is the C-terminal β-signal used for?

Answer 36

Insertion from C-terminus helps target it to BamA and initiate folding. (unlike alpha-helix inserted from N-term , while it's still being synthesised by the ribosomes)

Question 37

What is the role of BamA in outer membrane protein insertion?

Answer 37

Uses hybrid barrel model Opens lateral gate for insertion

Question 38

What is the insertion recognition signal in β-barrel TM proteins?

Answer 38

Located in the C-terminal strand > last strand to be synthesised but first to be inserted

Question 39

What is the insertion recognition signal in Alpha-helix TM proteins?

Answer 39

in the N-terminal hydrophobic sequence > inserted as it's still being synthesised

Question 40

Why does BamA have lateral gate opening?

Answer 40

the pairing between N- and C- terminal barrel strands are labile > can easily be opened to insert novel beta-barrels > exception in beta-barrels (usually they r strong)

Question 41

How can we extract membrane proteins? and why?

Answer 41

with detergents ! > req for high res analysis: x-ray, cryoEM, NMR after extraction we reconstruct the membrane

Question 42

Role of detergents in membrane ? how hard is it?

Answer 42

Solubilise the lipid and form micelles around proteins > usually membrane proteins readily denature with incorrect conditions so this process is difficult

Question 43

What are the characteristics of bicelles used in membrane protein studies?

Answer 43

Formed by mixing long-tail lipids with detergents Disc-shaped with a central planar bilayer Successful for aiding crystallization of membrane proteins

Question 44

What are nanodiscs in membrane protein studies?

Answer 44

Consist of a patch of 130-160 lipids Surrounded by membrane scaffolding proteins (MSPs) > inc stability > allow size control

Question 45

Hydrophilic domain and loops connecting the transmembrane element protrude from the membrane on extra/intracellular side r/f

Answer 45

true

Question 46

True or False A hydrophilic part is always responsible for the membrane interaction of the proteins

Answer 46

False, the hydrophobic part is responsible for this