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Y2 Biostructures > Membrane proteins > Flashcards

Membrane proteins Flashcards

1
Q

How are tightly bound membrane proteins solublised?

A

Use of detergent or organic solvent. Sometimes solutions with high ionic strength

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2
Q

Why are integral membrane proteins difficult to separate from the plasma membrane?

A

They interact with internal fatty acid tails.
Can be released by agents which compete for these non-polar interactions

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3
Q

How are peripheral membrane proteins bound to lipid heads?

A

Bound by electrostatic and hydrogen bond interactions.
Can be bound to surfaces of integral proteins

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4
Q

What types of hydrophobic lipid anchor are there?

A

Acylation
Prenylation
GPI lipid anchor

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5
Q

What is acylation?

A

Hydrophobic lipid anchor on the inner leaflet of plasma membranes. Same length as hydrocarbons in phospholipids. N-terminal methionine has to be removed to expose glycine. Amide binds to this.

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6
Q

What is prenylation?

A

Hydrophobic lipid anchor with a thioester link to cysteine on the C-terminus. This pattern is -Cys-a-a-X (a being an aliphatic amino acid). When something attaches to the C-terminus, aax is cleaved.

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7
Q

What are GPI anchored proteins comprised of?

A

Phosphate, inositol, mannose and ethanolamine
Hydrophobic pore, hydrophilic on the outside

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8
Q

Why aren’t GPI-anchored proteins used for transport across the membrane?

A

They don’t span the membrane

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9
Q

In membrane channels, why do residues on the outside of the pore tend to have ring structures?

A

Act as dampers between hydrophilic and hydrophobic residues

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10
Q

Why is hydrogen bonding important in β-barrel proteins?

A

It is energetically unfavourable for unpaired carbonyls to be in a hydrophobic environment, hydrogen bonding counteracts this

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11
Q

What happens when there is a hole in the plasma membrane?

A

Everything would diffuse out the membrane and polarity would be lost
Proton gradient would disappear

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12
Q

How is bacteriorhodopsin structured?

A

7 α-helicies form a tight bundle. These are mainly made up of non-polar residues. Retinal is covalently bonded and absorbs light.

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13
Q

Where is free energy of amino acids in bacteriorhodopsin scored?

A

α-helix in the membrane interior to water

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14
Q

How can alpha helix structures be predicted?

A

Balancing free energy transfers of amino acids with hydropathy plots.

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15
Q

Why cannot β-barrel structures be predicted with hydropathy plots?

A

They have alternating hydrophobic and hydrophilic residues, charges would balance out.

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16
Q

How can membrane permeability be calculated?

A

(diffusion coefficient X water-membrane partition coefficient) / membrane thickness

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17
Q

What does a basic channel comprise of?

A

Hydrophobic interface which protects the core from the membrane
Vestibule- where the compound is collected
Narrow pore with a selectivity filter

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18
Q

Describe the structure of aquaporins

A

Tetramer made of 4 individual subunits, each with its own pore- 6 hydrophobic helices each
Helices are tilted at 30° and twisted into a right handed bundle.
2-fold symmetry axis (inverted topology repeats), indicating ancient gene duplication events

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19
Q

How do aquaporins make sure one water moves through the channel?

A

Arginine electrostatically repulses protons.
If water moving through is uninterrupted, a proton conducting wire is formed.
NPA motifs in the middle prevent ions moving through
Asn76 and Asn192 form hydrogen bond donors which flip the water dipole oxygen up, hydrogen down. This creates a temporary break, preventing protons moving with it.

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20
Q

What was the first evidence for glucose transporters?

A

Rate of glucose transport capped after a certain concentration. Transport was shown to be saturable.

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21
Q

Outline the mechanism of GLUT1

A
  1. ATP is hydrolysed to pump glucose across the membrane
  2. A clear channel is formed, connecting two sides of the membrane
  3. Glucose movement is facilitated by inward and outward facing conformations- alternating access
  4. Membrane potential is exploited to move glucose across the membrane
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22
Q

Where do primary active transporters get their energy from?

A

Use a primary source of energy, e.g ATP

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23
Q

Where do secondary active transporters get their energy from?

A

Two different substances are coupled. One molecule moves along its concentration gradient

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24
Q

How much quicker is passive transport than active transport?

A

10^4-10^8 times.

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25
Q

What does the jardetzky model postulate?

A

Transporter with alternating access- 1 side open at a time. The cavity which recognises the substrate has high affinity. When a conformational change takes place, binding affinity is reduced to release the substrate to the other side of the membrane.
This requires energy

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26
Q

Explain what lac permease is

A

Secondary active transporter which couples lactose to a proton gradient. ATP synthase maintains the proton gradient

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27
Q

How did the 12 transmembrane helices of lac permease arise?

A

12 transmembrane helices arisen from a duplication and fusion of a 6 transmembrane helix

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28
Q

Describe lac permease structure

A

12 transmembrane helices
Kinked helices for alternating access
Central hydrophilic cavity for sugar binding site
Two halves separated by a loop between TM6 and TM7

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29
Q

Outline the lac permease pumping cycle

A
  1. A proton binds to the carboxyl group in the binding site outside.
  2. Lactose binds the high affinity binding site.
  3. The conformation changes and affinity for lactose drops, allowing it to be pumped against its concentration gradient.
    The proton dissociates from the carboxyl group
  4. Thermal motion brings the conformation back to the state open outside the membrane
30
Q

Describe the basic K+ channel domain structure

A

Tetramer
3 helices: 2 transmembrane, 1 short pore (protecting the membrane)
Loops coming off the pore helix point inwards and contain carbonyl groups which act as the selectivity filter for K+.

31
Q

Where are KcsA channels found?

A

Prokaryotic cell membranes

32
Q

Where are KvaP channels found?

A

Eukaryotic cell membranes

33
Q

How do potassium ion channels attract K+?

A

Helices are arranged so that the end of the channel is negatively charged to attract positive ions

34
Q

How big is the potassium ion channel selectivity filter?

A

3Å. Ions with a diameter larger than 1.5Å cannot move through as ions are hydrated, increasing their diameter

34
Q

How big is the potassium ion channel selectivity filter?

A

3Å. Ions with a diameter larger than 1.5Å cannot move through as ions are hydrated, increasing their diameter

35
Q

Why is it energetically favourable for K+ channels to let through K+, but not Na+?

A

The smaller the ion, the more negative the free energy.
After water molecules are stripped away from the ion, the desolvation energy is repaid with resolvation energy from forming hydrogen bonds. Na+ does not repay this resolvation energy

36
Q

How is the passage of ions eased through K+ channels?

A

In the selectivity filter, K+ ions form a queue which mimics energy of hydration. The binding is weakened by electrostatic repulsion between ions and energy taken up for the conformational change in the selectivity filter

37
Q

When conditions are voltage gated potassium channels open?

A

Low pH

38
Q

What parts of KcsA and KvaP are analogues?

A

S5 and S6 in KvaP are analogues of TM1 and TM2 in KcsA.
S6 has a Gly hinge, similar to TM2 in KscA
S1 to S4 forms a membrane embedded sensor, Similar to helix 4 which has 4 arginine residues and is joined to a linker attached to the pore

39
Q

How does KvaP open?

A

The S4-S3 paddle responds to voltage changes and if it is less positive outside, it pulls on the S4-S5 linker to open the channel

40
Q

Why is bacteriorhodopsin easy to assay?

A

It changes colour

41
Q

How is retinal bound to bacteriorhodopsin?

A

Covalently attached by a schiff base to Lys216

42
Q

Outline the mechanism when light hits bacteriorhodopsin

A
  1. Retinal absorbs light and the protein shifts from an all-trans form to 13-Cis form
  2. In the L intermediate, the proton on N is released and binds to Asp85 as it has a hydrophobic environment
  3. In the late M intermediate, helix F swings out and opens the cytoplasmic half channel
  4. The pore fills with water, making it more polar, allowing the proton from Asp 96 to bind to retinol. Asp96 has a high pKa which allows this
  5. The proton from Asp 85 moves out and the channel reopens. Helix F closes
43
Q

What is the largest class of cell-surface receptors?

A

GCPRs

44
Q

What is an inhibitor is the β2 adrenergic receptor?

A

Carazolol

45
Q

What are the main similarities between rhodopsin and the β2 adrenergic receptor?

A

Locations of 11-cis retinal and the carazolol binding site

46
Q

How are Gα and Gγ subunits anchored to the plasma membrane?

A

Covalently attached fatty acids

47
Q

How is GDP displaced from the Gα subunit?

A

Upon receptor binding, the nucleotide binding site of Gα opens and GDP can be replaced by GTP

48
Q

Why does Gβγ dissociate from Gα?

A

When GTP binds to Gα, its surface changes conformation so that it no longer has an affinity for Gβγ.
Adenylyl cyclase then converts ATP to cAMP

49
Q

Describe the structure of adenylate cyclase

A

12 membrane-spanning helices
Two large cytoplasmic domains forming the catalytic part
Water soluble

50
Q

How is a second level of rate amplification produced in adenylate cyclase?

A

Epinephrine binding increases the rate of cAMP production

51
Q

Outline the mechanism of the GTPase clock

A

Gα subunits have GTPase activity which hydrolyses its bound GTP to GDP and Pi. The GDP bound Gα reassociates with Gβγ

52
Q

How can a G protein be reset

A

Hormone dissociating from the receptor
Signalling cascade which phosphorylates serine and threonine in the C-terminal of the receptor
β-arrestin then binds to the phosphorylated receptor, making the G-protein less likely to be activated

53
Q

What are cone cells used for in vision?

A

Detecting bright light and colour

54
Q

What are rod cells used for in vision?

A

Dim light

55
Q

Describe rhodopsin

A

Found in membrane-enclosed sac-discs.
Made of opsin protein 11-cis retinal
Absorbs in the middle of the visible spectrum (500nm, green)
Gives an order of magnitude greater extinction coefficient than Trp

56
Q

Describe rhodopsin

A

GPCR with a polyene tail which absorbs light
Aldehyde groups form a schiff base with lys296 on helix 7
Retinal has a protonated schiff base in rhodopsin as it absorbs >440nm

57
Q

Why does retinal in rhodopsin absorb wavelengths >440nm?

A

Free retinal absorbs maximally at 370nm
Retinal has a protonated schiff base in rhodopsin as it absorbs >440nm

58
Q

How is the positive charge of retinal in rhodopsin accounted for?

A

The positive charge is compensated by glutamate on helix 2 (counter ion)

59
Q

How is a rhodopsin GPCR switched off?

A
  1. A conformational change takes place, catalyses displacement of GDP by GTP, promoting dissociation of the G-protein
  2. The α subunit regulates a phosphodesterase, causing ion channel closure and drop in cGMP
  3. Membrane becomes hyperpolarised, triggering neurotransmitter release
  4. The transductin binding site on the c-terminus is phosphorylated by rhodopsin kinase
    β-arrestin switches the whole system off
60
Q

How is a rhodopsin shift in absorption triggered?

A

Light hits rhodopsin (red), changing it from 11-cis retinal to all-trans retinal bathorhodopsin
The schiff base is deprotonated, causing a shift in absorption for metaphodopsin (bleached)

61
Q

What identity do blue photoreceptors have with red and green photoreceptors?

A

40%

62
Q

What identity do red and green photoreceptors have with each other

A

95%
3 amino acid difference
Green: AFA (ala, phe, ala)
Red: SYT (ser, tyr, thr)

63
Q

How are protein structures determined?

A

X ray scattering of protein crystals

64
Q

How are protein crystals structured?

A

Sparse networks of weak intermolecular interactions
Crystals are delicate as there are large gaps filled with buffer
Array of proteins in the same orientation

65
Q

What can spots from x-ray scattering tell us about protein strucutre?

A

The position of each spot tells us the amount of detail and arrangement of crystal planes
Amplitude of every diffraction spot tells us the types of atoms there

66
Q

What are the ideal conditions for crystallising a protein?

A

Abundant supply of protein
Isoforms of the species
Mutants for different stabilities and sensitivity profiles
Cleavable tags for simple and quick purification
95% protein purity
Homogenous sample
1-10mg/ml stable sample

67
Q

What method is usually used to purify membrane proteins

A

Bilayer is disrupted
If proteins are pulled straight out, they aggregate in water
Detergents are delicately designed to take membrane proteins straight out

68
Q

What properties do detergents have?

A

Water soluble surfactants. Alter surface tension
Amphiphiles, so are good at solubilising membrane components
More soluble in water than lipids
Most synthetic detergents have a polar headgroup and non-polar tail

69
Q

What is the critical micellular concentration?

A

Concentration that detergents self-associate into micelles. To maintain protein solubility, you should get above the CMC
Hydrophobic parts of the membrane protein are coated by detergent to protect it from aqueous solution

70
Q

How are protein samples prepared for cryo-EM?

A
  1. Sample is applied to a 3mm grid
  2. Blotting to remove extra liquid
  3. This is plunged into liquid ethane to freeze, giving buffer no time to crystalise.
  4. This is transferred to an electron microscope. The proteins are arranged in different orientations so a 3D picture can be taken
71
Q

What is a limitation of Cryo-EM?

A

Limited to large proteins (>500kDa)

Y2 Biostructures (5 decks)

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