Counterions and the bacteriorhodopsin proton pump

AbstractTheoretical and new experimental arguments are given to explain the reversal of photoelectric signals from purple membranes oriented and immobilized in gel due to the presence of TEMED. The continuous current induced by continuous illumination demonstrates a photoelement-like behaviour, the polarity of which is reversed by TEMED. The data render the counterion-collapse mechanism highly questionable

Optikai mikromanipuláció a biofizikában = Optical micromanipulation in biophysics

A projekt új lézercsipesz laboratórium kiépítését finanszírozta. Fő fejlesztés egy fény térmodulátorral (Spatial Light Modulator -SLM) felszerelt lézercsipesz megépítése. Ezzel tetszőlegesen sok csapda független egyidejű programozására van lehetőség. Új litográfiás berendezést is beszereztünk, ezzel mikrofluidikai eszközöket és integrált optikai elemeket készítünk. Az új laboratóriumban új típusú optikai mikromanipulációs kísérleteket végeztünk. Bonyolult alakú teszt objektumokkal összetett mozgásokat lehet megvalósítani. Négy típusú kutatást folytattunk: 1. A torziós manipulációs lehetőséget kihasználva DNS molekula csavarási tulajdonságait viszgáltuk. 2. A fotopolimerizációs struktúra építést és az új lézercsipeszt kihasználva új vizsgálati eszközöket készítettünk, mint mikroviszkozitásmérő, optikai mikromanipulátor. Modellrendszert alkottunk biológiai mozgások modellezésére: Kísérletileg kimutattuk és jellemeztük a hidrodinamikai szinkronizáció jeléenségét. 3. A folyadék mozgatásának fénnyel való vezérlését továbbfejlesztettük, a folyadék áramlási mintázatának fénnyel való változtatását oldottuk meg mikrofluidikai csatornában. 4. Integrált optikai elemeket készítettünk fotopolimerizációval mikrofluidikai alkalmazásra. Nagy érzékenységű interferometrikus szenzort készítettünk, ezt intermolekuláris reakciók jellemzésére, illetve optoelektronikai logikai áramkörök építésére alkalmaztuk. | The project supported the development of a new optical tweezers laboratory. The main development was the building of optical tweezers based on a Spatial Light Modulator (SLM). With this there is possibility to independently program an arbitrary number of optical traps. We also purchased a new photolythography device, this supportsthe building of microfluidics elements and integrated optical parts. In the new laboratory we performed novel optical manipulation experiments.We can realise complicated motions with test objects of complex shape. We worked on four types of experiments: 1. Using the possibility to rotate the trapped objects, we performed torsional manipulation experiments on DNA molecules. 2. Applying the photopolymerisation technique and the new optical tweezers we developed new experimental methods, like microviscosimeter, optical micromanipulator. We also created a model system to mimic biological motions. We experimentally demonstrated and characterised the phenomenon of hydrodynamic synchronisation. 3. We further developed the optical control of fluid flow: we realised the opticaal change of flow pattern in a microfluidics channel. 4. We built integrated optical elements for microfluidics applications. We built a high sensitivity interferometric sensor, and we used this to follow intermolecular interactions and to create optoelectronic logical circuit elements

Actinic Light-Energy Dependence of Proton Release from Bacteriorhodopsin

Measuring the light-density (fluence) dependence of proton release from flash excited bacteriorhodopsin with two independent methods we found that the lifetime of proton release increases and the proton pumping activity, defined as a number of protons per number of photocycle, decreases with increasing fluence. An interpretation of these results, based on bending of purple membrane and electrical interaction among the proton release groups of bacteriorhodopsin trimer, is presented

Buffer effects on electric signals of light-excited bacteriorhodopsin.

Buffers change the electric signals of light-excited bacteriorhodopsin molecules in purple membrane if their concentration and the pH of the low-salt solution are properly selected. "Positive" buffers produce a positive component, and "negative" buffers a negative component in addition to the signals due to proton pumping. Measurement of the buffer effects in the presence of glycyl-glycine or bis-tris propane revealed an increase of approximately 2 and a change of sign and a decrease to approximately -0.5 in the translocated charge in these cases, respectively. These factors do not depend on temperature. The Arrhenius parameters established from the evaluation of the kinetics indicate activation enthalpies of 35-40 kJ/mol and negative activation entropies for the additional signals. These values agree with those found by surface-bound pH-sensitive probes in the search of the timing of proton release and uptake. The electric signals were also measured in the case of D(2)O solutions with similar results, except for the increased lifetimes. We offer a unified explanation for the data obtained with surface-bound probes and electric signals based on the clusters at extracellular and cytoplasmic sites of bacteriorhodopsin participating in proton release and uptake

Photoexcitation of the O-Intermediate in Bacteriorhodopsin Mutant L93A

During the extended lifetime of the O-state in bacteriorhodopsin (bR) mutant L93A, two substates have been distinguished. The first O-intermediate (OI) is in rapid equilibrium with N and apparently still has a 13-cis chromophore. OI undergoes a photoreaction with a small absorbance change, positive charge transport in the pumping direction, and proton release and uptake. None of these effects was detected after photoexcitation of the late O (OII). The most likely interpretation of the effects seen is an accelerated return of the molecule from the OI- to the bR-state. However, with a lifetime ≈140 ms, the reaction cannot account for the observed high pumping efficiency of the mutant under continuous illumination. We suggest that OII corresponds to the O-intermediate with a twisted all-trans chromophore seen in the photocycle of wild-type bR, where the 13-cis OI-intermediate under the usual conditions does not accumulate in easily detectable amounts and, therefore, has generally been overlooked. Both the OI- and OII-decays are apparently strongly inhibited in the mutant

Non-proton ion release in purple membrane.

Large conductivity changes have been measured during the photocycle of bacteriorhodopsin in purple membrane. These phenomena were explained as being due to the occurrence of large-scale non-proton ion release. Here we show that these conductivity changes do not appear if the purple membrane is immobilized. We propose an alternative hypothesis that explains the presence of conductivity change in suspensions and their absence in gels, as well as several related effects suggesting that the observed conductivity changes are due to alteration of the polarizability of purple membrane during the photocycle