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Holography BR

Holography BR

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Published by: raalbe_autor on Dec 17, 2010
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Photorefractive Protein bacteriorhodopsin, BR , as a material forholographic data-recording
Rafael Alemañ
1
y María Gomariz
21
Grupo de Biomateriales. División de Óptica. Departamento de Ciencia deMateriales, Óptica y Tecnología Eectrónica, Universidad Miguel Hernández. C/ Avda. Universidad, s/n. Edif. Torrevaillo 03202-Elche, España.
2
División deMicrobiología, Departamento de Producción Vegetal y Microbiología, UniversidadMiguel Hernández. C/ Avda. Universidad, s/n. Edif. Torregaitán, 03202-Elche,España.
SUMMARY
The search of materials of biological originwith specific estates of potential technologicapplications whose structure and functionscome out from a process of adaptiveevolution experienced by the organisms inextreme environments, is one of the mostactive fields in the modern biophysics.In short, the protein bacteriorhodopsin, actslike a protonic bomb whose photocycle hasdemonstrated interesting implications in thetechnologies of optic storage of informationby means of holographic methods.In the present work some results of theinvestigation undertaken by our group areexposed, consistent mainly in the study of the influence that working conditions (pH,state of aggregation of the environment,intensity and time of illumination, etc.) haveover photophysical and photochemical statesof bacteriorhodopsin as well as in thetracking of the possible presence of bacteriorhodopsin proteins andxantorhodopsin, in the salines of Santa Pola(Alicante, Spain).1.
INTRODUCTION
 For the bioengineering of the XXI century,learning from the benefits demonstrated bythe adaptive structures generated by naturalselection, and using proteins as functionalunits incorporating them in the moderntechnological devices, are two of the mostimportant challenges.In this sense, molecules as photoactiveproteins are able to transform the lightdirectly into an electric signal [1]. Thisprocess implies the formation of an electricdipole and occur by changing proteinspectrum.Chlorophyll (in vegetables and bacteria) andrhodopsin (in animals and bacteria) are themost extended photosensitive biomoleculesin our planet whose role as converters of solar energy make them very well-known.The basic structure of the rhodopsin consistsof retinal molecules connected to proteins(opsins) with a specific structure by meansof a Schiff base. In almost all the naturalrhodopsins, the cromophore is the portion of retinal in the complex retinal-protein due toits high molar extinction coefficient, itsexcellent quantum yield and its shortisomerization time. The proteins and retinalthat separately absorb in the UV range whencombining, shift their spectrum of absorption to the visible region. It isnecessary to highlight that the proteincomplexes of the retinal transport ionsthrough the membranes, while chlorophyllproduces separations of electric charges.1
 
In 1971 there was discovered a complexprotein derivate of the retinal in the externalwrapping of one of the archaeobacteriaspecies, inhabitant of strongly salineenvironments [2]. Named bacteriorhodopsin(from now on, BR) due to their similaritywith the visual rhodopsin of the eye, it usesthe energy of the solar light to manage anionic transport of hydrogen through thecellular membrane creating this way anelectric potential that allows the synthesis of adenosín triphosphate (ATP).This is the better studied example of proteinphotoactive and it presents very diversetechnical applications, which in turn havegenerated an important research activity [3].BR, forming what is known as purplemembrane in archaeobacteria, allows thosemicroorganisms to obtain metabolic energystarting from the light in extreme conditionsof high salinity, low oxygen concentrationand high solar radiation [4]. In stagnatedwaters some kinds of bacteria (asSalinibacter ruber) contain in theirmembrane another protein together withretinal, called xantorrhodopsin (XR) thatalso works as a protonic pump [5].2.
THE RHODOPSIN EVOLUTION
The evolution of photosensitivemolecules as rhodopsins, it is tied mostly tothe process of adaptation of living beings tothe environments with diffused light. Thefirst vertebrates that appear in the fossilrecord lived in shallow waters where lightarrived muffled. Many of the animals thatnowadays inhabit such atmospheres possessrhodopsin molecules with a maximum of absorption around 500 nanometres, whatbelongs together with the ghastly intensitiesin the twilight or in situations of veiled light.In these molecules the maxima of absorption in certain wavelengths can beexplained by means of the substitution of 15amino acids in 12 different places. It seemsthat these fifteen amino acids weresubstituted numerous times in theevolutionary course of the rhodopsins. Thefact that the maximum of absorption in theancient rhodopsins locates around the 500nm, would explain the reason that theircurrent descendants evolved –at least up to18 different times– in order to comprise awider range, of among 480-525 nm.And although the dominat tendencyamong researchers has pointed to awardingthese adaptive changes to a selectivepressure of darwinian type, there are alsoother authors who regard the neutralisttheory of molecular evolution as a betterexplanation [6]. Anyway, it seems beyondall doubt that the statistical tests of positiveselection are difficult to obtain in amolecular level. The adaptive phenomenain the evolutionary course of the rhodopsins,must be elucidated through mutagenicanalysis using ancient pigments related withthem.Only this way we will be able tounderstand how the photosensitivemolecules have ended up acquiring theabsorption spectrum that they in factpossess. In nature diverse visual pigmentsadapted to different environmentalbrightness exist. And to determine themolecular mechanisms involved in theadaptive selection of such pigments,weshould first identify the aminoacidic changesthat are potentially important in themodification of the wavelength for themaximum absorption. In fact the possibilityof using directed-mutagenesis methods andgrowths of cellular fabrics, makesphotosensitive pigments an ideal model forthe study of adaptive mechanisms at themolecular level.2
 
 
Fig. 1. Rhodopsin molecule.
Graphicrecreation of the multiple-helix structure of arhodopsin molecule3.
EVOLUTIONARY ADAPTATIONAND TECHNOLOGICALOPTIMIZATION
 Living organisms thoroughly take benefitfrom the recognition of molecularconformations, what simultaneosulyestablishes remarkable similarities anddifferences with the inert systems. One of the basic component of an electronic device,for example, is a switch; and photosensitiveproteins, in turn, can be considered in fact assomething similar to a switch. Therhodopsin, let us say, absorbs a photon and itunchains a sequence of biochemicalreactions that end in a nervous impulse, asimilar performance to that of aphototransistor.Nevertheless, along evolution, nature hasappealed to a probe-and-error method tosolve by means of suitable moleculesorganic problems that are similar to thosethat we find in inorganic molecules takenadvantage to carry out logical functions, of exchange or management of data.For that reason, when we think in biophysicsabout the technologic application of biological materials, we must deal with threefundamental questions:1)
 
Which are the elementary physicalfeatures of the process that we areapproaching?2)
 
Is there some biological system thatpossesses those same features or carryout similar functions with anequivalent purpose?3)
 
Of all the varying adaptativas that theevolution has been able to generate,according to the different colonizedenvironments, which is the best onethat fits our demands?Since our purpose was the search of photosensitive materials for the digitalstorage of information by means of opticprocedures, it seemed logical to think of themost versatile photosensitive proteins. Andparticularly in the BR, since it has a greatstability against chemist as thermaldegradation, its photophysic behaviorresembles that of the chlorophyll (but withan inferior yield that does not overcome 15% on converting light energy in chemicalenergy), besides being reversible andtherefore subjected to repeated usesThe BR is a protein located in some regionsof the bacterial membrane collectivelydenominated purple membrane (MP).Forming a hexagonal crystalline net in theMP, the molecules of BR are notdenaturalized until the 80 ºC (or evensometimes until the 160 ºC), and they retaintheir cromophoric estates in a pH range from00,0-11,0. Their advantages are:It shows an extreme simplicity of thephotosynthetic system, with the protonictransfer carried out by a single molecule.Thanks to the specific constitution of thebacterial cellular wall and the packaging of BR in the MP, the bacteria survive underextreme environmental conditions (high3

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