Bacteriorhodopsin The Purple Membrane Protein


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Bacteriorhodopsin


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







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




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