8 - Bacteriorhodopsin

Question 1

What does bacteriorhodopsin do?

Answer 1

It uses light to pump protons across the membrane

Question 2

Which amino acid is important for the proton gradient?

Answer 2

Asp - unprot in hydrophilic, prot at edge (more hydrophobic)

Question 3

How is the proton gradient established in mitochrondria?

Answer 3

Through cytochrome proteins

Question 4

What was the controversy about ATP synthase in the 1960s?

Answer 4

People thought it worked due to conformational change, but it actually works through proton gradient

Question 5

What experiment showed that ATP synthase used a proton gradient?

Answer 5

1. ATP synthase and bacteriorhodopsin were constituted in lipid vesicles
2. Added ADP and light, and saw ATP being produced

Question 6

What bacteria is bacteriorhodopsin from?

Answer 6

Haloarchaea

Question 7

Where do haloarchaea live?

Answer 7

In salt flats

Question 8

Why do haloarchaea have a purple color?

Answer 8

From the retinal (absorb light)

Question 9

What is another name for retinal?

Answer 9

Vitamin A

Question 10

How long is the cycle from trans to cis in retinal?

Answer 10

~1 ms

Question 11

What happens when retinal absorbs light?

Answer 11

It goes from trans to cis

Question 12

What is attached to the retinal?

Answer 12

Lys

Question 13

What happens to the Schiff base during the reaction?

Answer 13

It gets deprotonated (pumped across membrane)

Question 14

What was the first retinal binding protein discovered?

Answer 14

Rhodopsin in the eye

Question 15

What is optogenetics?

Answer 15

Use light to open ion channels on neurons

Question 16

What are some other members of the bacteriorhodopsin family?

Answer 16

Chloride pumps (halorhodopsin), phototaxis (sensory rhodopsins), ion channels (channel rhodopsins), and marine bacteria proton pump (proterorhodopsin)

Question 17

What is the structure of bacterorhodopsin?

Answer 17

7 transmembrane helices, retinal in middle

Question 18

What are the steps of the bacteriorhodopsin pathway?

Answer 18

1. Use light to isomerize to retinal 13-cis (K/L)
2. Protonate equilibrium between Schiff base and Asp85 (M1)
3. Switch and release proton to outside (M2/M2’)
4. Reprotonation of Schiff base by Asp96 (N)
5. Reprotonation of Asp96, reisomerization of retinal to all trans (O)
6. Deprotonation of Asp85 (back to bR)

Question 19

What direction does the retinal move when it isomerizes?

Answer 19

Away from the direction it pumps

Question 20

Where is Asp212 located and what does it do?

Answer 20

Located next to Asp85, and helps stabilize protonated Schiff base

Question 21

True or false: bR has functional water

Answer 21

True: they aid in H-bonding, which is important for the intermediates

Question 22

What happens when the retinal isomerizes?

Answer 22

The Schiff base is put into a hydrophobic environment, dropping the pKa (want to deprot)

Question 23

How does the Schiff base in bR get deprot?

Answer 23

The Asp85 (deprot) is not stabilized, so its pKa changes and it gets prot

Question 24

What is the energy of a photon?

Answer 24

50 kcal/mol

Question 25

What happens when Asp85 gets prot?

Answer 25

Arg82 (prot) is not stabilized by negative charge, so it swings over to Glu194-Glu204 pair

Question 26

What is the Glu-Glu pair similar to?

Answer 26

Asp proteases

Question 27

What are the initial states of the Glu-Glu pair?

Answer 27

One prot, one unprot

Question 28

What happens when Arg82 interacts with the Glu-Glu pair?

Answer 28

Glu204 deprot (pKa drops, stabilized by new positive charge on Arg82)

Question 29

What happens to TM6 when Asp85 gets prot?

Answer 29

It moves, allowing water to interact with Asp96

Question 30

How does TM6 get moved?

Answer 30

Methyl group pushes on Trp182, acts as a lever to push helix

Question 31

How is prot Arg82 stabilized in br?

Answer 31

Through water molecules

Question 32

What is unique about Arg partial positive charge?

Answer 32

It is distributed over many atoms

Question 33

What happens when Asp96 is exposed to water?

Answer 33

The pKa of Asp96 (prot) drops (stabilized by H-bonds), thus reprot the Schiff base

Question 34

What happens when the Schiff base is reprot?

Answer 34

It triggers the thermal reisomerization to all trans

Question 35

What happens when retinal reisomerizes?

Answer 35

1. TM6 moves back to close cavity and force water back out, which reprot Asp96 by picking up a proton from the cell 2. Asp85 deprot (due to prot Schiff base), Glu204 reprot, and Arg82 swings back to Asp85

Question 36

What is the general overview of the bR reaction?

Answer 36

Series of protonation changes, directed by pKa modulations and small movements in the protein

Question 37

How can bR be turned into a Cl- pump?

Answer 37

By mutating Asp85 to Thr85

Question 38

How does Thr85 interact with the mutated bR?

Answer 38

It is neutral, and chloride can enter

Question 39

How does the mutated bR work?

Answer 39

When retinal reisomerizes, the Cl- follows NH+ on Schiff base, and Cl- can enter cytoplasm when TM6 moves to allow water in