8 - Bacteriorhodopsin Flashcards
What does bacteriorhodopsin do?
It uses light to pump protons across the membrane
Which amino acid is important for the proton gradient?
Asp - unprot in hydrophilic, prot at edge (more hydrophobic)
How is the proton gradient established in mitochrondria?
Through cytochrome proteins
What was the controversy about ATP synthase in the 1960s?
People thought it worked due to conformational change, but it actually works through proton gradient
What experiment showed that ATP synthase used a proton gradient?
- ATP synthase and bacteriorhodopsin were constituted in lipid vesicles
- Added ADP and light, and saw ATP being produced
What bacteria is bacteriorhodopsin from?
Haloarchaea
Where do haloarchaea live?
In salt flats
Why do haloarchaea have a purple color?
From the retinal (absorb light)
What is another name for retinal?
Vitamin A
How long is the cycle from trans to cis in retinal?
~1 ms
What happens when retinal absorbs light?
It goes from trans to cis
What is attached to the retinal?
Lys
What happens to the Schiff base during the reaction?
It gets deprotonated (pumped across membrane)
What was the first retinal binding protein discovered?
Rhodopsin in the eye
What is optogenetics?
Use light to open ion channels on neurons
What are some other members of the bacteriorhodopsin family?
Chloride pumps (halorhodopsin), phototaxis (sensory rhodopsins), ion channels (channel rhodopsins), and marine bacteria proton pump (proterorhodopsin)
What is the structure of bacterorhodopsin?
7 transmembrane helices, retinal in middle
What are the steps of the bacteriorhodopsin pathway?
- Use light to isomerize to retinal 13-cis (K/L)
- Protonate equilibrium between Schiff base and Asp85 (M1)
- Switch and release proton to outside (M2/M2’)
- Reprotonation of Schiff base by Asp96 (N)
- Reprotonation of Asp96, reisomerization of retinal to all trans (O)
- Deprotonation of Asp85 (back to bR)
What direction does the retinal move when it isomerizes?
Away from the direction it pumps
Where is Asp212 located and what does it do?
Located next to Asp85, and helps stabilize protonated Schiff base
True or false: bR has functional water
True: they aid in H-bonding, which is important for the intermediates
What happens when the retinal isomerizes?
The Schiff base is put into a hydrophobic environment, dropping the pKa (want to deprot)
How does the Schiff base in bR get deprot?
The Asp85 (deprot) is not stabilized, so its pKa changes and it gets prot
What is the energy of a photon?
50 kcal/mol
What happens when Asp85 gets prot?
Arg82 (prot) is not stabilized by negative charge, so it swings over to Glu194-Glu204 pair
What is the Glu-Glu pair similar to?
Asp proteases
What are the initial states of the Glu-Glu pair?
One prot, one unprot
What happens when Arg82 interacts with the Glu-Glu pair?
Glu204 deprot (pKa drops, stabilized by new positive charge on Arg82)
What happens to TM6 when Asp85 gets prot?
It moves, allowing water to interact with Asp96
How does TM6 get moved?
Methyl group pushes on Trp182, acts as a lever to push helix
How is prot Arg82 stabilized in br?
Through water molecules
What is unique about Arg partial positive charge?
It is distributed over many atoms
What happens when Asp96 is exposed to water?
The pKa of Asp96 (prot) drops (stabilized by H-bonds), thus reprot the Schiff base
What happens when the Schiff base is reprot?
It triggers the thermal reisomerization to all trans
What happens when retinal reisomerizes?
- TM6 moves back to close cavity and force water back out, which reprot Asp96 by picking up a proton from the cell
- Asp85 deprot (due to prot Schiff base), Glu204 reprot, and Arg82 swings back to Asp85
What is the general overview of the bR reaction?
Series of protonation changes, directed by pKa modulations and small movements in the protein
How can bR be turned into a Cl- pump?
By mutating Asp85 to Thr85
How does Thr85 interact with the mutated bR?
It is neutral, and chloride can enter
How does the mutated bR work?
When retinal reisomerizes, the Cl- follows NH+ on Schiff base, and Cl- can enter cytoplasm when TM6 moves to allow water in
