Characterization of the Proton-Transporting Photocycle of Pharaonis Halorhodopsin

AbstractThe photocycle of pharaonis halorhodopsin was investigated in the presence of 100mM NaN3 and 1M Na2SO4. Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR′ state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide

Programozható optoelektronikus tömbprocesszorok (POAC) és alkalmazásaik = Programmable opto-electric array processors (POAC) and their applications

Kidolgoztunk és megépítettünk egy hordozható programozható opto-elektronikus analogikus CNN számítógépet (Laptop-POAC), amelyet céltárgyak felismerésére és követésére alkalmaztunk. A POAC (programmabble optical analogic array computer) magprocesszora egy új típusu optikai korrelátor, amelyben bakteriorhodopsin (BR) filmet alkalmaztunk dinamikus (újraírható, többszörösen olvasható, törölhető) holografikus anyagként. Ez az optikai CNN implementáció egyesíti az optikai számítógépek nagy sebességét, rendkívűl nagy práhuzamosságát (milliós nagyságrendű csatornaszám) és a nagymértű templatek alkalmazhatóságát a CNN eszköszök rugalmas programozhatóságával. Különös jellemzője ennek az optikai tömbprocesszornak az, hogy a programozó templateket vagy egy kétdimenziós akuszto-optikai eltérítővel (ez esetben 64x64 pixel méretű templateket használtunk), vagy egy VCSEL lézer mátrixszal valósíthatjuk meg. A bemenő képeket 600x800 pixel felbontású folyadékkristályos mikromegjelenítővel vittük be. Meghatároztuk a jelenleg beszerezhető kulcselemekkel elérhető maximális felbontás és a sebesség korlátait. Ezáltal kimutatttuk, hogy a kispárhuzamosságú elektronikus adatátvitel (továbbá a létező CCD, CMOS és vizuális CNN chipek felbontás/sebesség) korlátai miatt 2D-s optika utófeldolgozásra van szükség és lehetőség. Új és hatékony céltárgy felismerő és több tárgy kvázi-egyidejű követésére alkalmas algoritmust dolgoztunk ki. Mérésekkel bizonyítottuk a berendezés és az algoritmusok hatékonyságát. | A portable programmable opto-electronic analogic CNN computer (Laptop-POAC) has been built and used to recognize and track targets. Its kernel processor is a novel type of high performance optical correlator based on the use of bacteriorhodopsin (BR) as a dynamic holographic material. This optical CNN implementation combines the optical computer's high speed, high parallelism (1 000 000 channel) and large applicable template sizes with flexible programmability of the CNN devices. A unique feature of this optical array computer is that programming templates can be applied either by a 2D acousto-optical deflector (up to 64x64 pixel size templates) incoherently or by VCSEL arrays. Input images are fed-in by a LCD-SLM of 600x800 pixel resolution. Evaluation of trade-off between speed and resolution is given. Novel and effective target recognition and multiple-target-tracking algorithms have been developed for the POAC. Tracking experiments are demonstrated. In the present model a video-speed CCD camera is recording the correlograms, however, later a CNN-UM chip and a high-speed CMOS camera will be applied for combined optical/electro-optical post-processing. An optical CNN template- and algorithm-library has been developed to solve a great variety of image processing tasks. It seems to be an important future work to expand this library

Biológiai membránok elektromos térszerkezetének vizsgálata = Investigation of the charge structure of biological membranes

A pályázat célja a biológiai membránok fizikai tulajdonságainak vizsgálata volt, különös tekintettel a töltéseloszlás és - ezzel összefüggésben - az elektromos térszerkezet funkcionális szerepére. Modellobjektumunk a protonpumpáló bakteriorodopszin (bR) fehérjét kristályos rendben tartalmazó bíbormembrán. Kutatásainkat és a velük kapcsolatos módszertani fejlesztőmunkát három téma köré csoportosítottuk: 1. A molekulán belüli elektromos töltéseloszlás funkcionális leírása 2. A határfelületi vízréteg szerepe a fehérjék szerkezetében és működésében 3. A bR nemlineáris optikai tulajdonságainak jellemzése Eredmények: 1.Megmutattuk, hogy az általunk kifejlesztett módszer a molekula működését kísérő szerkezetváltozásokat érzékenyen jellemzi, és lehetőséget teremt az elektromos töltésátrendeződések funkcionális értelmezésére. 2.Rámutattunk, hogy a semleges sók anionjainak tulajdonított Hofmeister-effektus a fehérje-víz határfelületi réteg szerkezetváltozásaival magyarázható. Elméletünk elsőként ad teljeskörű kvalitatív leírást a 120 éves problémakörre. 3.Integrált optikai módszerekkel az eddigieknél érzékenyebben detektáltuk a bR molekulában - a lokális elektromos tér változásai következtében ? fellépő abszorpció- és törésmutató-változásokat. Megmutattuk, hogy a molekula gerjesztését követő spektrális változások alkalmasak fénykapcsolásra és -modulálásra. Az eredmények gyakorlati alkalmazása az optoelektronikában és a bioszenzorikában várható. | In the framework of the project, we investigated some physical properties of biological membranes, with special respect to the physiological role of the electric charge distribution (hence the electric field structure). Our model object was purple membrane, containing the paradigmatic proton pumping molecule bacteriorhodopsin (bR) in a quasi crystalline structure. Our research work and the related methodological developments were centered round three topics: 1.Functional description of the intra-molecular charge distribution 2.The role of interfacial water in the structure and function of proteins 3.Characterization of the nonlinear optical properties of bR Results: 1.We showed that our method represents a sensitive test for the characterization of structural changes associated to molecular function, and allows interpretation of charge rearrangements. 2.We pointed out that the Hofmeister effect, attributed to the anions of neutral salts, can be interpreted according to structural changes in the protein-water interface. Our theory gives a full-spectrum qualitative description, solving a 120-years old puzzle. 3.Using integrated optical (IO) techniques, we could sensitively characterize the nonlinear optical properties of bR films, and showed that IO light switching and modulation can be performed based on the light-induced spectral changes of bR. Practical utilization of the results is expected in optoelectronics and sensorics

Membrán fehérjék szerkezete és működése közti kapcsolat vizsgálata = The study of the connection between the structure and function of membrane proteins

A 2005 januárjában kezdődőtt négy éves OTKA támogatással a membrán fehérjék szerkezete és működése közti kapcsolatok tisztázására folytatott kutatás két fő irányba haladt. Az egyik a membrán fehérjék és itt főleg a fényhajtotta retinál fehérjék működésével kapcsolatos kutatásokra összpontosított. Ennek során nemcsak a jól ismert retinál fehérjék a bakteriorodopszin, vagy halorodopszin, hanem újonnan felfedezett fehérjék, mint például a xantorodopsin és leptospheria rodopszin működését is tanulmányoztuk, abszorpciókinetikai mérésekkel. A retinál fehérjék kutatásával kapcsolatosan, az OTKA pályázat támogatásával, nyolc cikket közöltünk. Hasonló kinetikai méréseket végeztünk fotoszintetikus rekciócentrumok és szén nanocsövek keverékén, ami ígéretes biotechnologiai anyagnak bizonyúl. A reakciócentrumokkal kapcsolatban két OTKA támogatta cikkünk jelent meg A másik fő kutatási irány a fehérjék, membránok és élő sejtek morfológiai és mechanikai vizsgálata atomerő mikroszkóppal. Természetes és mesterséges membránokon is végeztünk méréseket. Amíg különböző letapogatási technikákkal nagyfelbontású képet alkottunk a tanulmányozott objektumok felszínéről, addig erőméréssel sikerült meghatározni a tanulmányozott minta Young moduluszát és ennek a változását különböző külső körülmények között. Egy teljesen új technikát dolgoztunk ki retinál fehérjék konformációváltozásának atomerőmikroszkópos megfigyelésére. Az elmúlt négy évben ezekkel a mérésekkel kapcsolatban nyolc OTKA által támogatott cikkelt közöltünk. | Beginning in 2005 the four years OTKA supported study of the relation between the structure and function of membrane proteins was conducted in two main directions. One research direction concentrates on the function of the membrane proteins and mainly on the function of the light activated retinal proteins. In this study not only the well known bacteriorhodopsin and halorhodopsin, but some newly discovered proteins, such as the xantorhodopsin and leptospheria rhodopsin were studied by absorption kinetic measurements. Related to this research, eight papers supported by the OTKA were published. Similar kinetic measurements were performed on the mixture of photosynthetic reaction centers and carbon nanotubes. This material is considered as a promising biotechnological material. The results of the reaction center research were published in two OTKA supported papers. The second main research direction was the morphological and mechanical study of the proteins, membranes and living cells by atomic force microscopy. We performed measurements on both artificial and natural membranes. With different scanning techniques high resolution images of the studied object surface were recorded. With force measurement it was possible to determine the Young?s module of the sample and its dependence from the external conditions. A new technique was developed to study the conformational change of the retinal proteins with the atomic force microscope. During the OTKA supported four years, related to this field, eight papers were published

Interaction of cysteine-rich cationic antimicrobial peptides with intact bacteria and model membranes

Antimicrobial peptides are small proteins that exhibit a broad spectrum of antimicrobial activity. Their chemical structure allows them to interact (attach and insert) with membranes. The fine details about this interaction and their mode of action are not fully clarified yet. In order to better understand this mechanism, we have performed in situ atomic force microscopy studies using two types of nodule specific cysteine-rich NCR peptides on Escherichia coli bacteria and on natural purple membrane. On intact bacteria, both NCR247 and NCR335 caused increase in the surface roughness, indicating the damage of the bacterial cell envelope. In case of the tightly packed purple membrane, it is clear that the peptides prefer to disrupt the border of the disks indicating a strong lipid preference of the interaction. These results verify the concept that the first target of NCR peptides is probably the bacterial cell envelope, especially the lipid matrix

Effect of Antimicrobial Peptide-Amide: Indolicidin on Biological Membranes

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity is intensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here an in situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membrane of dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependent interaction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purple membrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the border of membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool in the study of indolicidin-membrane interaction

Elasto-mechanical properties of living cells

The possibility to directly measure the elasticity of living cell has emerged only in the last few decades. In the present study the elastic properties of two cell lines were followed. Both types are widely used as cell barrier models (e.g. blood-brain barrier). During time resolved measurement of the living cell elasticity a continuous quasi-periodic oscillation of the elastic modulus was observed. Fast Fourier transformation of the signals revealed that a very limited number of three to five Fourier terms fitted the signal in the case of human cerebral endothelial cells. In the case of canine kidney epithelial cells more than 8 Fourier terms did not result a good fit. Calculating the correlation between nucleus and periphery of the signals revealed a higher correlation factor for the endothelial cells compared to the epithelial cells

Addressing the optimal silver content in bioactive glass systems in terms of BSA adsorption

Bioactive glasses doped with silver are aimed to minimize the risk of microbial contamination, and therefore, the influence of silver on the bioactive properties is an intense investigated task. However, the information related to the role played by silver, when added to the bioactive glass composition, on the biocompatibility properties is scarce. This aspect is essential as long as the silver content can influence the blood protein adsorption onto glass surface, affecting thus the material biocompatibility. Therefore, from the perspective of the biocompatibility standpoint, the finding of an optimal silver content in a bioactive glass is an extremely important issue. In this study, silver doped bioactive glasses were prepared by melt-derived technique, which eliminates the pores influence in the protein adsorption process. The obtained glasses were characterized by X-ray diffraction, UV-vis, X-ray Photoelectron (XPS) and Fourier Transform Infrared (FT-IR) spectroscopy, and afterwards they were investigated in terms of protein adsorption. Both UV-vis and XPS spectroscopy revealed the presence of Ag+ ions in all silver containing samples. By increasing the silver content, metallic Ag0 appears, the highest amount being observed for the sample with 1 mol% AgO2. Electron Paramagnetic Resonance measurements evidenced that the amount of spin labeled serum albumin attached on the surface increases with the silver content. The results obtained by analyzing the information derived from Atomic Force Microscopy and FT-IR measurements indicate that the occurrence of metallic Ag0 in the samples structure influences the secondary structure of the adsorbed protein. Based on the results derived from the protein response upon interaction with the investigated glass calcium-phosphate based system it was determined the optimal silver oxide concentration for which the secondary structure of the adsorbed protein is similar with that of the free one. This concentration was found to be 0.5 mol%

Carbon nanotubes quench singlet oxygen generated by photosynthetic reaction centers

Photosensitizers may convert molecular oxygen into reactive oxygen species (ROS) including, e.g., singlet oxygen (1O2), superoxide anion (O2-•), and hydroxyl radicals (•OH), chemicals with extremely high cyto- and potential genotoxicity. Photodynamic ROS reactions are determinative in medical photodynamic therapy (cancer treatment with externally added photosensitizers) and in reactions damaging the photosynthetic apparatus of plants (via internal pigments). The primary events of photosynthesis take place in the chlorophyll containing reaction center protein complex (RC), where the energy of light is converted into chemical potential. 1O2 is formed by both bacterial bacteriochlorophylls and plant RC triplet chlorophylls in high light and if the quenching of 1O2 is impaired. In plant physiology, reducing the formation of the ROS and thus lessening photooxidative membrane damage (including the RC protein) and increasing the efficiency of the photochemical energy conversion is of special interest. Carbon nanotubes, in artificial systems, are also known to react with singlet oxygen. To investigate the possibility of 1O2 quenching by carbon nanotubes in a biological system, we studied the effect of carbon nanotubes on 1O2 photogenerated by photosynthetic RCs purified from purple bacteria. 1,3-Diphenylisobenzofuran (DPBF), a dye responding to oxidation by 1O2 with absorption decrease at 420nm was used to measure 1O2 concentrations. 1O2 was produced either from a photosensitizer (methylene blue) or from triplet photosynthetic RCs and the antioxidant capacity of carbon nanotubes was assessed. Less 1O2 was detected by DPBF in the presence of carbon nanotubes, suggesting that these are potential quenchers of this ROS. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim