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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
What does the jardetzky model postulate?
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
26
Explain what lac permease is
Secondary active transporter which couples lactose to a proton gradient. ATP synthase maintains the proton gradient
27
How did the 12 transmembrane helices of lac permease arise?
12 transmembrane helices arisen from a duplication and fusion of a 6 transmembrane helix
28
Describe lac permease structure
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
29
Outline the lac permease pumping cycle
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
Describe the basic K+ channel domain structure
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
Where are KcsA channels found?
Prokaryotic cell membranes
32
Where are KvaP channels found?
Eukaryotic cell membranes
33
How do potassium ion channels attract K+?
Helices are arranged so that the end of the channel is negatively charged to attract positive ions
34
How big is the potassium ion channel selectivity filter?
3Å. Ions with a diameter larger than 1.5Å cannot move through as ions are hydrated, increasing their diameter
34
How big is the potassium ion channel selectivity filter?
3Å. Ions with a diameter larger than 1.5Å cannot move through as ions are hydrated, increasing their diameter
35
Why is it energetically favourable for K+ channels to let through K+, but not Na+?
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
How is the passage of ions eased through K+ channels?
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
When conditions are voltage gated potassium channels open?
Low pH
38
What parts of KcsA and KvaP are analogues?
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
How does KvaP open?
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
Why is bacteriorhodopsin easy to assay?
It changes colour
41
How is retinal bound to bacteriorhodopsin?
Covalently attached by a schiff base to Lys216
42
Outline the mechanism when light hits bacteriorhodopsin
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
What is the largest class of cell-surface receptors?
GCPRs
44
What is an inhibitor is the β2 adrenergic receptor?
Carazolol
45
What are the main similarities between rhodopsin and the β2 adrenergic receptor?
Locations of 11-cis retinal and the carazolol binding site
46
How are Gα and Gγ subunits anchored to the plasma membrane?
Covalently attached fatty acids
47
How is GDP displaced from the Gα subunit?
Upon receptor binding, the nucleotide binding site of Gα opens and GDP can be replaced by GTP
48
Why does Gβγ dissociate from Gα?
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
Describe the structure of adenylate cyclase
12 membrane-spanning helices Two large cytoplasmic domains forming the catalytic part Water soluble
50
How is a second level of rate amplification produced in adenylate cyclase?
Epinephrine binding increases the rate of cAMP production
51
Outline the mechanism of the GTPase clock
Gα subunits have GTPase activity which hydrolyses its bound GTP to GDP and Pi. The GDP bound Gα reassociates with Gβγ
52
How can a G protein be reset
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
What are cone cells used for in vision?
Detecting bright light and colour
54
What are rod cells used for in vision?
Dim light
55
Describe rhodopsin
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
Describe rhodopsin
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
Why does retinal in rhodopsin absorb wavelengths >440nm?
Free retinal absorbs maximally at 370nm Retinal has a protonated schiff base in rhodopsin as it absorbs >440nm
58
How is the positive charge of retinal in rhodopsin accounted for?
The positive charge is compensated by glutamate on helix 2 (counter ion)
59
How is a rhodopsin GPCR switched off?
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
How is a rhodopsin shift in absorption triggered?
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
What identity do blue photoreceptors have with red and green photoreceptors?
40%
62
What identity do red and green photoreceptors have with each other
95% 3 amino acid difference Green: AFA (ala, phe, ala) Red: SYT (ser, tyr, thr)
63
How are protein structures determined?
X ray scattering of protein crystals
64
How are protein crystals structured?
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
What can spots from x-ray scattering tell us about protein strucutre?
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
What are the ideal conditions for crystallising a protein?
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
What method is usually used to purify membrane proteins
Bilayer is disrupted If proteins are pulled straight out, they aggregate in water Detergents are delicately designed to take membrane proteins straight out
68
What properties do detergents have?
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
What is the critical micellular concentration?
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
How are protein samples prepared for cryo-EM?
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
What is a limitation of Cryo-EM?
Limited to large proteins (>500kDa)

Y2 Biostructures (5 decks)

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