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Membrane proteins: Bacteriorhodopsin

Bacteriorhodopsin is a light-driven proton pump that was first found in halophilic archaea. Recently it was shown that a homologous group of proteins called proteorhodopsins are widly distributed in aquatic bacteria. It allows the cells to harvest the energy of the sun light for phototrophic growth. In contrast to the photosynthesis of endosymbiontic chloroplasts in plants and previously known groups of photosynthetic bacteria no electron transport is involved. Therefore bacterio- and proteorhodopsin-mediated phototrophic growth results in “proton transport phosphorylation, in contrast to the “electron transport phosphorylation” of the classical photosynthesis. However, both processes involve integral membrane proteins and result in an electrochemical gradient across a membrane which powers the ATPsynthase.  Theoretical concepts include:
  •  the differences of integral membrane proteins and soluble proteins;
  •  the structure of membranes and their diverse functions in transport, signaling, and energy metabolism;
  •  bacteriorhodopsin-mediated proton transport (proteorhodopsin is not in the textbooks yet);
  •  the “classical” photosynthesis of plants and several bacterial groups.
 Proposed book chapters NC 11, 19 and VV 20, 24.

Reset PyMOLBacteriorhodopsin
Bacteriorhodopsin (BR) is a relatively small membrane protein (26 kDa). As all membrane proteins, it has been particularly challenging to structural analysis, due to difficulties in the process of purification. Membrane proteins, contrary to non-membrane proteins, expose their non-polar (hydrophobic) residues to the exterior.
BR functions as a pump of protons from the cytoplasm to the extracelullar space, in order to create a proton gradient. Afterwards, protons enter the cell again favourably, and the cell takes advantage of that by coupling to a reaction that synthesizes ATP. The energy required by BR is provided by green light. At the end of the process, the outcome is that the cell transformed energy from light into ATP, the energetic currency of the cell.
Launch PyMOL

Load 1c3w
fetch 1C3W
Show cartoon
show cartoon
hide lines
BR is a trimer. Structurally each monomer is mainly constituted by 7 trans-membrane alpha-helices, connected by 3 intracellular and 3 extracellular loops. C-terminus is located in the cytoplasm while the N-terminus is in the periplasm. Load the PDB structure into PyMOL (1C3W) and try to identify those elements. Use a cartoon representation.

Create objects
create retinal, resn RETcreate waters, resn HOHcreate lipids, resn LI1+SQU+RET+HOH+LI1+SQU
Show objects
hide linesshow sticks, retinal + lipidsshow spheres, waterscolor red, retinalcolor orange, lipids
In this module we will focus on the use of the PyMOL command line. The first task is to create different objects for the different components in the structure: protein, retinal, water molecules and lipids. To find out the name of the heteroatoms, it is convenient to display the sequence. From the external GUI, "Display-> Sequence" and then "Display->Sequence Mode->Residue Codes". Scroll to the end and find out the names for the non-aminoacid groups. Retinal is RET, water HOH and the lipid fragments LI1 and SQU. Create individual objects, by using the "resn" (residue name) keyword and choose an appropriate view: protein as cartoon (hide lines), lipids and retinal as sticks, waters as spheres. Color retinal red and lipids orange.

Show hydrophobics yellow
hide everything, lipidscolor whiteselect hydrophobics, resn ala+leu+val+ile+pro+phe+met+trpselect hydrophilics, not hydrophobics and not resn HOH+LI1+SQU+RETcolor yellow, hydrophobicscolor cyan, hydrophilics
Show ribbons
hide everythingshow ribbon
Show spheres - hydrophobics
hide spheres
show spheres, hydrophobics
Show spheres - hydrophilics
hide spheresshow spheres, hydrophilics

Show charged
select charged, resn asp+glu+arg+lyscolor magenta, charged
After the general overview, let's take a look at the particularities of the protein. First let's study the distribution of hydrophobic and hydrophilic residues. Hide the lipids, show the protein in sticks and color in yellow the hydrophobic residues (ala, leu, val, ile, pro, phe, met, trp) and in cyan the hydrophilic ones. Is it possible to identify patterns in the distributions? Where do you see hydrophilic ones preferently, in the interior or at the exterior? What is the composition of the loops?

Switch to a ribbon representation.

Switch alternatively hydrophobics and hydrophilics to a sphere representation. How much from the backbone can you still see in both cases?
What about aromatic residues? Do they distribute equally or do they concentrate in certain regions? With all this information, could one infer that we are actually talking about a transmembrane protein?

Finally, select the charged residues and color them in magenta. Observe how they are arranged to facilitate the circulation of the proton.

Delete objects
delete retinal + waters + lipidsLoad 1dze
fetch 1DZE

Align proteins
align 1DZE, 1C3Wcenter 1C3WShow ribbons
hide everythingshow ribbon

Show retinals
select c-retinal, 1DZE and resn RETselect t-retinal, 1C3W and resn RETcolor green, 1C3Wcolor red, 1DZWshow sticks, c-retinal + t-retinal
To get some insight into the biological function, it is useful to compare this structure with another one in which the retinal has already changed conformation (from trans to cis).
For this, delete the extra objects we created.

Load 1DZE using the PDB loader plugin.

Align it to the first structure and center the scene.

Show only a ribbons representation of the protein.
Color the structure with t-retinal in green and the other one in red.

Show both retinal molecules in sticks, by creating two separate selections. Observe how tiny  the change is, an isomerization caused by the absorbtion of green light. Now let's explore the impact of this change in the rest of the residues, which finally permits BR to function.

Display Lys-216
select lys216_trans, resi 216 and 1C3Wselect lys216_cis, resi 216 and 1DZEshow sticks, lys216_trans + lys216_cislabel name ca and byres lys216_trans, "%s - %s"%(resn, resi)

Display Asp-85
select asp85_trans, resi 85 and 1C3Wselect asp85_cis, resi 85 and 1DZEshow sticks, asp85_trans + asp85_cislabel name ca and byres asp85_trans, "%s - %s"%(resn, resi)
Show waters
select waters, resn HOHshow spheres, watersset sphere_scale, 0.6color red, resn HOH and 1c3wcolor white, resn HOH and 1dze

Show additional residues
select additional_residues, resi 57+82+194+204show sticks, additional_residueslabel name ca and byres additional_residues, "%s - %s"%(resn, resi)
In the ground state retinal is bound to the amino group of Lys-216. In addition, a number of water molecules that can be found in the interior of the channel form part of a network that stabilizes the structure. When the retinal isomerizes it causes a change in Lys-216, and a series of hydrogen bond reorientations happens all the way down to Asp-96. This is reflected in a cascade of conformational changes that eventually starts the transport of a proton from the cytosol to the outside. We cannot see protons here because they are not present in a crystal structure, but we can observe some of the tiny conformational changes that occur.
Start by selecting Lys-216 for cis and trans states, show them in sticks and label one of them with residue number and residue name.

Do the same for Asp-85.

Show water molecules in spheres. Color in red the molecules belonging to the t-retinol structure and in white the others. Observe how they are also involved in the network. Scale the spheres to 60%. This is the original network that is broken when the conformational change of the retinal takes place.
The bond between Lys-216 and retinal is a protonated Schiff's base. At this point, this base transfers the proton to Asp-85 and then reorients away from the channel to prevent reprotonation. It has been shown that residues Tyr-57, Arg-82, Glu-194, and Glu-204 also mediate the transport of the proton. Show these residues in sticks and label them.

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