Bacteriorhodopsin

Introduction

• Bacteriorhodopsin (BR) is a retinal protein molecule found in the photosynthetic system of a salt-marsh bacterium called Halobacterium salinarium.

• In its native form, the BR molecule is located in a cell membrane commonly called the purple membrane (PM).

• Within the bacterial cell, BR is critical to the survival of the organism in an oxygen-deficient environment, as the BR molecules function as light-driven proton pumps which transport protons across the cell membrane.

• This generates a proton gradient which in turn produces an electrochemical potential used by the organism to synthesize adenosine triphosphate (ATP).

• Effectively, BR is used by the bacterium to directly convert sunlight into chemical energy.

• The absorption of light also initiates a photocycle in the BR molecule which accompanies the transportation of protons.

• The characteristics and effects of this photocycle make it a potentially useful material for development as an optically sensitive film that is self-developing and erasable.

• A tremendous advantage of BR's organic nature is that it readily lends itself to genetic engineering, which allows the generation of genetic variants that may possess significantly different optical characteristics.

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)

salt-marsh

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

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

Halobacterium halobium

• Resides in hypertonic environments (shallow waters of San Francisco bay)

• Diatomic oxygen is plentiful = OXPHOS• Diatomic oxygen unavailable = LIGHT HARVESTING

– Achieved by Bacteriorhodopsin, a 26 KD protein with all-trans-retinal attached

• LIGHT IS CONVERTED DIRECTLY INTO A H+ MOTIVE FORCE

F0F1 ATPaseH+

+ + + + + + +

- - - - - - - - -

H+

ATP

hv

Retinal is bound to Lysine

H+ pumping

Photocycle• The energy trapping

conformational transition occurs within one ps

• Deprotonation and reprotonation steps are characterized by intermediates which contain distinct absorption spectra

• One full turn of the cycle occurs within a ms

• Hence each step has a characteristic colour

• Every sec, 50 protons are exported

• One question remains…how does the light induced isomerization of this protein drive H+ translocation

Bacteriorhodopsin is a Dynamic, Transmembrane Protein

Several transmem spanning helices3 Tyr hold the BR in place

Retinal sits at the junction separating the two“half” channels

Notice the Bacteriorhodopsin is surrondedby Asp and Arg

Nanotechnology properties

• (a) Photochromic properties - Bacteriorhodopsin as material for optical information recording: Biotechnological applications on the basis of the colour change between purple and yellow (long living intermediate M) is the basis for using of bacteriorhodopsin for optical information recording. The technique has advanced such that it could be used as a safety feature on chipcards.

•  • Isomerization from all-trans to 13-cis is the first occurrence after

the photochemical excitation of bacteriorhodopsin, and this causes significant transient shifts in the absorption spectrum. In addition to the isomerization change, deprotonation of the chromophoric group is observed. In the L to M transition, a proton from the Schiff base nitrogen group is transferred to asp85, and this deprotonation causes a drastic blue shift of the absorption to 410 nm. The photochromisim of bacteriorhodopsin is dominated by the intermediate which has the longest lifetime: this forms a “bottleneck” in the photocycle.

•  

• Upon acidification, a blue membrane is formed which has a significant different photocycle. The formation of a 9-cis retinal-containing state is observed, and this is thermally stable. In contrast, 13-cis retinal is isomerized by the bacteriorhodopsin molecule to all-trans retinal at room temperature, and the isomerization of 9-cis retinal is not catalyzed. This pathway opens the route to long-term storage materials based on bacteriorhodopsin.

 • Three types of photochromic changes in

bacteriorhodopsin have been described which enable different applications. The first is the photochromic shift between the B and M states, and this is used mainly for optical processing tasks. The second is photoerasable data storage using 9-cis-containing states of the blue membrane or suitably modified BR-variants. And last, permanent photochromic changes obtained through two-photon absorption in bacteriorhodopsin are suitable for long-term data storage.

Preparation of Bacteriorhodpsin films:

• Optical films are prepared from bacteriorhodpsin by polymer embedding. Optically clear, water-soluble polymers are suitable for this purpose (e.g polyvinylalcohol, gelatin). The film formation is carried out by making the polymers with PMs and additives in aqueous solution, this being cast on a glass support. The water is generally removed by drying in air, but the films may also be sealed with a second glass plate.

(b) Photoelectric properties:

• Upon illumination, a photovolatage up to 250 mV per single bacteriorhodopsin layer is generated. Triggered by the absorption of a photon, the bacteriorhodopsin molecule undergoes a series of very rapid molecular changes, The proton released through the outer proton half channel may be either transferred to outer medium. As all of the bacteriorhodopsin molecules in a single PM patch are oriented in the same direction, the voltage generated over a single membrane is independent of the number of active molecules, but the proton current generated is proportional to the light intensity. The photovoltage generated can be easily measured by embedding the PM layer between two transparent electrodes.

• Preparation of oriented PM layers: The Langmuir-Blodgett technique is used for preparation of single layer of PM.  

4. Applications• Some of the bionanotechnological applications that are

anticipated include: • Ultrarapid optical data acquisition with parallel processing

capabilities;• extreme high density holographic three-dimensional data and

image storage;• Unique optical filters; • Highly sensitive photodetectors and sensors; • Color-transitioning security links for forgery and counterfeit

prevention. • Adapting natural membranes or engineering alterations in

those structures would have significant advantages over the artificial membranes that are currently employed.

• The naturally derived membranes would be biodegradable, eliminating the necessity for the disposal of products that would be toxic or recalcitrant to decomposition. Another major advantage would be the broad utility of the membranes with a variety of bioengineered bacteriorhodopsin molecules.

• APPLICATIONS IN OPTICS: To explore and utilize for development of photonic materials based on bacteriorhodpsin and its mutants for many different applications in optics include – holography, object recognition, interferometry, optical memory, real time information processing, detection of small vibrations, novelity filters, optical switches and many others.

• APPLICATIONS IN ELECTRONICS: Bacteriorhdopsin is a natural photoelectric generator and each step of the bacteriorhodopsin photocycel is accompanied by generation of a corresponding photocurrent. Several photoelectrical and electro-optical effects include modulation of transistor amplification with ultrahigh speed.