Bacteriorhodopsin The Purple Membrane Protein Mike Goodreid CHEM*4550 seminar.

BacteriorhodopsinBacteriorhodopsin

The Purple Membrane Protein

Mike GoodreidCHEM*4550 seminar

Outline of PresentationOutline of Presentation

Introduce Bacteriorhodopsin (BR)History of its structural analysisStructural features of the proteinMechanism of actionEnergy involved in action

Source of BRSource 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 What is the purple membrane?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?So what does BR do?

BR functions as a proton pumpLong 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 ProtonsPhotons 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 Milestones in BR Structural DeterminationDetermination

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 Nobel Prize in Chemistry (1988)(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 ExperimentHartmut’s Experiment

FindingsFindings

Could get protein crystallizationCrystals were too small and disordered to

determine tertiary structureResults uncommon because

– BR is a very stable protein– BR forms a 2D lattice in vivo and in vitro

(later)

19901990

Henderson et al. use cryo-crystallography to study BR

Crystallization occurredFirst instances of structural determinationHowever, some areas of the protein could

not be resolved

1990 First structure of BR1990 First structure of BR

First structures of BR from side and top/bottom

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

Paradigm shift in crystallization of membrane proteins

Use Cubic Lipid Phase MatrixFirst complete structural determination of

BR

Intro to CLPIntro 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 phasebehaviour occurs (say goodbye to micelles and bilayers)

Seeding and FeedingSeeding 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 FeedingSeeding 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)

ResultsResultsHexagonal crystals from MO bicontinuous lipid phase lead to complete structural determination of BR(3.7 Angstrom resolution)

BR gene expressionBR gene expression

786 nt structural gene13 AA precursor sequence

+248 AA in mature BR

+1 AA (D) at C-terminal sequenceNo intervening sequencesNo prokaryotic promoter (yet?)

Brp has role in retinal synthesis from beta-caroteneBlh has a similar role(?)

Structural Features of BRStructural Features of BR

Extracellular matrix

Cytosol

H+ | |V

Structural InfoStructural 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-Are There Any Highly-conserved Residues?conserved 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.

PhotocyclePhotocycle

A lesson in pushing protons

Photocycle of BR begins with absorption of a photon withwavelength of 550 nm. All-trans retinal13-cis retinal

1313 |CHO

All-trans retinal (blue)Carbon 13 (red)

Photocycle of BR begins with absorption of a photon withwavelength of 550 nm. All-trans retinal13-cis retinal

1313 |CHO

13-cis retinal (blue+cyan)Carbon 13 (red)

Photocycle (K)Photocycle (K)

Extracellular matrix

Cytosol

H+ | |V

K

H

H

PRS H

cis

Photocycle (L)Photocycle (L)

Extracellular matrix

Cytosol

H+ | |V

KL

H

H -Partial retinal relaxation-Subtle changes in protein conformation

PRS H

cis

Photocycle (M)Photocycle (M)

Extracellular matrix

Cytosol

H+ | |V

LM

H

H -K216 (schiff base deprotonated)-D85 picks up proton (perhaps via H2O intermediate)-Proton lost from PRS

PRS

cis

Photocycle (N)Photocycle (N)

Extracellular matrix

Cytosol

H+ | |V

MN

H

H

-D96 deprotonated-K216 picks up proton

PRS

cis

Photocycle (O)Photocycle (O)

Extracellular matrix

Cytosol

H+ | |V

NO

H

H

-Retinal reisomerizes back to All-Trans -D96 reprotonated from cytosol

PRS

H

Photocycle (final step)Photocycle (final step)

Extracellular matrix

Cytosol

H+ | |V

OK

H

H

-D85 deprotonated -PRS reprotonated-back to square 1 until another proton isomerizes the All-trans retinal

PRS

H

Basic BiophysicsBasic Biophysics

And now for something completely different

Thermodynamics of TransportThermodynamics 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-20JSince 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 poweredCation pump

-Facilitate environmentalcleanup of heavymetals

-Cheap, easy way of accumulating protons:

-Industry-Fuel cell cars

ReferencesReferencesLanyi, 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. USA93, 14532-14535

Nollert, P. Qiu, H., Caffrey, M., Rosenbusch, J.P., and Landau, E.M. (2001)FEBS Lett. 504, 179-186