Optical and electric signals from dried oriented purple membrane of bacteriorhodopsins R. Tóth-Boconádi a , A. Dér a , L. Keszthelyi a,b, ⁎ a Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, P. O. B. 521, H-6701, Hungary b KFKI Research Institute for Particle and Nuclear Physics, Hungarian Academy of Sciences, Budapest P.O.B. 49, H-1525, Hungary abstract article info Article history: Received 11 June 2010 Received in revised form 16 December 2010 Accepted 19 December 2010 Available online 28 December 2010 Keywords: Bacteriorhodopsin mutants Quasi-continuous illumination M intermediate Blue excitation Lifetimes All the intermediates of the bacteriorhodopsin photocycle are excitable with light of suitable wavelength. This property might regulate the activity in the cells when they are exposed in the nature to high light intensity. On the other hand this property is involved in many applications. In this study the ground state and M intermediate of dried oriented samples of wild-type bacteriorhodopsin and its mutant D96N were excited with 406 nm laser flashes. Substantial M populations were generated with quasi-continuous illumination. The decay of the absorption of M intermediate had three components: their lifetimes were very different for laser flash and quasi-continuous illuminations in cases of both bacteriorhodopsin species. The optical answer for the excitation of M intermediate had a lifetime of 2.2 ms. Electric signals for M excitation had large fast negative components and small positive components in the 100 μs time domain. The results are expected to have important implications for bioelectronic applications of bacteriorhodopsin. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The possible application of the dried oriented purple membrane layers containing the light sensitive protein, bacteriorhodopsin (BR) [1–3] in different electronic and optical tools became evident in the last years [4–11]. The photocycle of BR in dried samples is restricted: it reaches the M intermediate for light excitation, stops there and finally returns to ground state with a lifetime of seconds. The most important characteristic of these samples is their stability: they maintain this restricted function for years or, with other term, not damaged during 10 6 –10 7 photon induced cycles. It is well known that the intermediates of the BR photocycle can be excited by light [12–22]. This process is important in the life of the cells. As pointed out first in [12] the blue illumination of the M intermediate short-cuts the photocycle, i.e. blocks the proton pumping activity, this way protecting the cells from excessive chemical potential in case of high illumination. The special features of intermediate excitation, like wavelength or lifetime, are useful in some applications: for example the Q form arising from excited O via another intermediate P may serve as a memory element [23]. The M intermediate is especially important because its excitation terminates the photocycle, i.e. reestablishes the BR ground state in short time as investigated in the usual wet samples [12–16,22]. In view of the urgency of a recent boom in bioelectronic applications using dry BR films [5–11], it seemed to be worthwhile also to know the time needed for this process for dried oriented purple membrane layers. Hence, we investigated the optical and electric responses of the M intermediate of the wild-type bacteriorhodopsin (WTBR) and its mutant D96N in dried oriented samples. First the ground state was excited and investigated. It turned out in absorption measurements at around 400 nm (the characteristic wavelength of M decay) that the lifetimes and amplitudes of the three decay components depend on the strength and duration of illumination. Studying the optical answer for M excitation, absorption changes of ms duration emerged during the reestablishment of the ground state. In the electric responses for M excitation two negative components appeared in the μs time range and a third positive component with a lifetime of around 100 μs. 2. Materials and methods Dried oriented samples of purple membrane separated from strain R 1 M 1 of Halobacterium salinarum and strain L-33 containing the mutant D96N (supplied by J. K. Lanyi) were prepared as described in [1]. In order to obtain substantial M populations the samples were illuminated with quasi-continuous (Q-C) light (quasi-continuous means continuous illumination for some s) from a 200 W tungsten lamp filtered for wavelengths above 500 nm, or by a 1 mJ flash of an excimer laser driven dye laser (Lambda Physik, EMG 101 MSC, Göttingen, Germany) with dye Rhodamine 6 G, emitting at 570 nm. The M concentration and the absorption changes for M excitation were determined using a Bioelectrochemistry 81 (2011) 17–21 ⁎ Corresponding author. Institute of Biophysics, Biological Research Centre, Hungar- ian Academy of Sciences, Szeged, P. O. B. 521, H-6701, Hungary. Tel.: +36 30 9194685; fax:+36 62433133. E-mail address: kl@brc.hu (L. Keszthelyi). 1567-5394/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2010.12.003 Contents lists available at ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem