Bacteriorhodopsin The Purple Membrane Protein

Bacteriorhodopsin

• The Purple Membrane Protein

Outline of Presentation

• Introduce Bacteriorhodopsin (BR)

• History of its structural analysis

• Structural features of the protein

• Mechanism of action

• Energy involved in action

Source of BR

• Archaebacteria Halobacteria Salinarium are the source of bacteriorhodopsin

• They are halophilic bacteria (found in very salty water e.g. Great Salt Lake)

What is the purple membrane?

• The purple membrane patches are areas on the membrane where BR is concentrated

• BR absorbs light @ 570 nm (visible green light)

• Red and Blue light is reflected, giving membrane its purple colour

So what does BR do?

• BR functions as a proton pump

• Long story short: protons are pumped one at a time from the inside of the cell to the outside

• Photons react with a bound retinal group causing conformational change in BR

Photons for Protons

• Bacteriorhodopsin takes energy from photons

• This energy is converted and creates a proton gradient by pumping protons outside the cell

• Protons are allowed back into the cell by an ATP synthase

• In a nutshell: Photons are used to power the cell

Milestones in BR Structural Determination

• In order to assess the structure and mechanism of BR, or any membrane protein, we really need to understand its tertiary structure by X-ray crystallography

• BUT, membrane proteins don’t crystallize easily

Nobel Prize in Chemistry (1988)

• Hartmut Michel

• First to crystallize BR in 1980

• Contribution to determination of structure of a photosynthetic reaction center earned him a Nobel Prize

Hartmut’s Experiment

Findings

• Could get protein crystallization

• Crystals were too small and disordered to determine tertiary structure

• Results uncommon because

• BR is a very stable protein
• BR forms a 2D lattice in vivo and in vitro (later)

1990

• Henderson et al. use cryo-crystallography to study BR

• Crystallization occurred

• First instances of structural determination

• However, some areas of the protein could not be resolved

1990 First structure of BR

1996: E.M Landau & J.P. Rosenbusch

• Paradigm shift in crystallization of membrane proteins

• Use Cubic Lipid Phase Matrix

• First complete structural determination of BR

Intro to CLP

Seeding and Feeding

• Purple membrane patches (or BR monomers) diffuse into the CLP

• Addition of Sorensen salt increases curvature of the CLP’s membranes

Seeding and Feeding

• Protein separates into planar domains (crystal formation)

• Mature crystals co-exist with BR depleted cubic phase

• Hydration (dilution of Sorensen salt solution) reverses the crystallization process (crystals dissolve back into CLP matrix)

Results

BR gene expression

• 786 nt structural gene

• 13 AA precursor sequence

• +248 AA in mature BR

• +1 AA (D) at C-terminal sequence

• No intervening sequences

• No prokaryotic promoter (yet?)

Structural Features of BR

• Cytosol

Structural Info

• 7 TM helices

• Forms a homotrimer

• Homotrimers aggregate to form the purple membrane

• Stability of trimer by:

• G113, I117, L48
• Most stability comes from surrounding lipids

Are There Any Highly-conserved Residues?

Photocycle

• A lesson in pushing protons

Photocycle (K)

• Cytosol

Photocycle (L)

• Cytosol

Photocycle (M)

• Cytosol

Photocycle (N)

• Cytosol

Photocycle (O)

• Cytosol

Photocycle (final step)

• Cytosol

Basic Biophysics

• And now for something completely different

Thermodynamics of Transport

• Energy of a photon:

• E=hc/lambda

• let lambda = 550 nm

• Ephoton=3.61x10^(-19) J

References