Bacteriorhodopsin
The Purple Membrane Protein
Mike Goodreid
CHEM*4550 seminar
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
First structures of BR from side and top/bottom
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
CLP matrix (bicontinuous cubic phase)
Involves -high lipid content
monoolein
(1-monooleoyl-rac-glycerol, C18:1c9, = MO)
-aqueous pores that
penetrate membrane
-proteins embedded
At high concentrations of lipids, more complex phase
behaviour occurs (say goodbye to micelles and bilayers)
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
Hexagonal crystals from MO
bicontinuous lipid phase lead to
complete structural determination of BR
(3.7 Angstrom resolution)
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?)
Brp has role in retinal synthesis from beta-carotene
Blh has a similar role(?)
Structural Features of BR
Cytosol
H+
|
|
V
Extracellular matrix
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 Highlyconserved Residues?
You’d better believe it!
L. Brown, 2001:
-Upon BLASTing the H. Salinarium BR, found very high homology
among all BR from a number of different Halobacterium
-Around the K216 schiff base, there is no deviation in AA
composition for a good 4.5 Angstroms
-This type of analysis shows the entire retinal binding pocket is
highly conserved. Therefore, MANY of the AAs in BR are
structurally and/or catalytically important. SDM is a useful tool for
validating this statement.
Photocycle
A lesson in pushing protons
1313 |
All-trans retinal (blue)
Carbon 13 (red)
CHO
Photocycle of BR begins with absorption of a photon with
wavelength of 550 nm. All-trans retinal13-cis retinal
1313 |
13-cis retinal (blue+cyan)
Carbon 13 (red)
CHO
Photocycle of BR begins with absorption of a photon with
wavelength of 550 nm. All-trans retinal13-cis retinal
Photocycle (K)
Cytosol
K
H
cis
H+
H |
|
V
PRS H
Extracellular matrix
Photocycle (L)
Cytosol
KL
H
cis
H+
|
H
|
V
PRS H
Extracellular matrix
-Partial retinal relaxation
-Subtle changes in protein
conformation
Photocycle (M)
Cytosol
LM
H
H+
|
|
V
cis
H
PRS
Extracellular matrix
-K216 (schiff base deprotonated)
-D85 picks up proton (perhaps
via H2O intermediate)
-Proton lost from PRS
Photocycle (N)
Cytosol
MN
H+
|
H
|
V
cis
H
PRS
Extracellular matrix
-D96 deprotonated
-K216 picks up proton
Photocycle (O)
Cytosol
NO
H
H+
|
H
|
V
H
PRS
Extracellular matrix
-Retinal reisomerizes back
to All-Trans
-D96 reprotonated from cytosol
Photocycle (final step)
Cytosol
OK
H
H+
|
H
|
V
PRS H
Extracellular matrix
-D85 deprotonated
-PRS reprotonated
-back to square 1 until
another proton isomerizes
the All-trans retinal
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
Energy req’d to move H+
/\G=RTln([H+out]/[H+in])
-zF/\psi
let: H+out=10,000 H+in,
T=295K
/\G=3.75E-20(J/H+) - zF/\psi
let: Vm=-60mV (an estimate)
/\G=(3.75(E-20) – 9.61E(-21)) J/H+
/\G = + 4.7E-20J
Since Ephoton>/\G, we can see that the photon is
sufficiently energized to move the proton
What promise does BR hold?
Bioengineering:
-Scaffold for a light powered
Cation pump
-Facilitate
environmental
cleanup of heavy
metals
-Cheap, easy way of
accumulating protons:
-Industry
-Fuel cell cars
References
Lanyi, J.K. (2001) Biochemistry (Moscow) 66, 1477-1482
Brown, L.S. (2001) Biochemistry (Moscow) 66, 1546-1552
Dunn, R., McCoy, J., Simsek, M., Majumdar, A., Chang, S.H., Rajbhandary, U.L.,
and Khorana, H.G..(1981) Proc Natl Acad Sci USA.78, 6744-6748
Jagannathan, K., Chang, R., and Yethiraj, A. (2002) Biophys J 83, 1902-1916
Peck, R.F., Echavarri-Erasun, C., Johnson, E.A., Ng, W.V., Kennedy S.P., Hood, L.,
DasSarma, S., and Krebs, M.P. (2001) J Biol Chem. 23, 5739-5744
Landau, E.M.and Rosenbusch, J.P. (1996) Proc. Natl. Acad. Sci. USA
93, 14532-14535
Nollert, P. Qiu, H., Caffrey, M., Rosenbusch, J.P., and Landau, E.M. (2001)
FEBS Lett. 504, 179-186