Aspartic acid

ABSTRACT The photocycle of dried bacteriorhodopsin, pretreated in a 0.3 M HCI solution, was studied. Some properties of this dried sample resemble that of the acid purple suspension: the retinal conformation is mostly all-trans, 15-anti form, the spectrum of the sample is blue-shifted by 5 nm to 560 nm, and it has a truncated photocycle. After photoexcitation, a K-like red-shifted intermediate appears, which decays to the ground state through several intermediates with spectra between the K and the ground state. There are no other bacteriorhodopsin-like intermediates (L, M, N, 0) present in the photocycle. The K to K' transition proceeds with an enthalpy decrease, whereas during all the following steps, the entropic energy of the system decreases. The electric response signal of the oriented sample has only negative components, which relaxes to zero. These suggest that the steps after intermediate K represent a relaxation process, during which the absorbed energy is dissipated and the protein returns to its original ground state. The initial charge separation on the retinal is followed by limited charge rearrangements in the protein, and later, all these relax. The decay times of the intermediates are strongly influenced by the humidity of the sample. Double-flash experiments proved that all the intermediates are directly driven back to the ground state. The study of the dried acid purple samples could help in understanding the fast primary processes of the protein function. It may also have importance in technical applications.

INTRODUCTION

Bacteriorhodopsin (BR), a light driven proton pump found in the cell membrane of Halobacterium salinarum was first isolated some 30 years ago (Blaurock and Stoeckenius, 1971). The studies concentrating on the structure and function of this protein revealed many details, beginning with the light absorption mechanism of the retinal bound to a lysine, to the final steps of proton translocation across the membrane (Lozier et al., 1975; Ebrey, 1993; Lanyi and Varo, 1995; Lanyi 2000). Dark adaptation of the protein preparation leads to a thermal equilibrium with the retinal in two different conformations: 40% all-trans, 15-anti, and 60% 13-cis, 15-syn form. Upon prolonged illumination the 13-cis retinal isomerizes to all-trans, forming the light-- adapted BR (Konishi and Packer, 1976; Scherrer et al., 1987).

After light excitation, BR undergoes a photocycle with several spectrally different intermediates (K, L, M, N, O) (Lozier et al., 1975; Gergely et al., 1997). The intermediates have complex kinetic properties (Varo and Lanyi, 1990; Lozier et al., 1992; Ludmann et al., 1998b) outlined in different models. Between pH 4 and 9, the function of the BR can be described with a model containing reversible reactions in a single photocycle (Varo and Lanyi, 1990; Ames and Mathies, 1990; Lozier et al., 1992; Ludmann et al., 1998b). At ~pH 9.5, which is the pK^sub a^ of the proton release complex, BR exists in two states with presumable protonated and deprotonated glutamic acid (Balashov et al., 1996; Richter et al., 1996). In this pH region, the possibility of the two parallel photocycles is reasonable.

Crystallographic studies resulted at 1.55 Angstrom resolution structure of the protein (Subramaniam, 1999; Luecke et al., 1999b). The structure of several photocycle intermediates have also been resolved (Edman et al., 1999; Luecke et al., 1999a; Vonck, 2000; Sass et al., 2000).

When pH is changed from neutral to acidic, several spectrally different species appear (Mowery et al., 1979; Varo, and Lanyi, 1989; De Groot et al., 1990). At pH below 2, the proton acceptor Asp^sup 85^ is protonated, which leads to different photocycles depending on the ion content of the bathing solution (Varo and Lanyi, 1989; Tokaji et al., 1997). When the solution contains sulfate but no chloride ions, the photocycle of the "acid blue BR" has a K-like and an L-like intermediate (Var6 and Lanyi, 1989). In the presence of chloride ions, the photocycle of the "acid purple BR" contains mostly red-shifted intermediates. Compared with BR, the decay of the excited state to a K-like intermediate is slower in this case (Logunov et al., 1996). After a K-like intermediate, an O-like one was observed (Varo and Lanyi, 1989). Later, it was shown that the photocycle also has an N-like intermediate (Tokaji et al., 1997). Fourier transform (FT) Raman studies revealed that the acid purple BR contains mostly all-trans retinal (Kelemen et al., 1999). The photocycle of acid purple BR was compared with that of halorhodopsin, using time-resolved Fourier transform infrared (FTIR) spectroscopy (Mitrovich et al., 1995)

Water has an important role in the function of BR, as was revealed by studies on dehydrated samples. During light-- adaptation, the 13-cis to all-trans transition of the retinal is increasingly hindered with decreasing humidity; at ambient humidity (P/P^sub o^ = 0.5) it is almost totally abolished (Korenstein and Hess, 1977a; Vard and Keszthelyi, 1983). The photocycle of the dried sample stops at intermediate M and no proton transport occurs (Korenstein and Hess, 1977b; Varo and Keszthelyi, 1983). The dehydration of the sample hinders the conformational change of the protein (Varo and Eisenstein, 1987). The photocycle terminates at intermediate M,, with the protein still in the extracellular conformation (Betancourt and Glaeser, 2000; Dencher et al., 2000), and returns back to ground-state BR after a multiexponential decay (Ganea et al., 1997). The kinetics of the photocycle are strongly dependent on the water content of the sample (Korenstein and Hess, 1977b; Varo and Keszthelyi, 1983; Ganea et al., 1997). FTIR studies revealed the importance of the bound water in the structure and function of BR (Maeda et al., 2000).

On electrically anisotropic samples containing oriented BR molecules, photoelectric response signals could be measured (Bamberg et al., 1981; Keszthelyi and Ormos, 1983; Der et al., 1985). In the pH range of 4 to 9, the electrogenicity of the intermediates was determined, which provided information about the proton translocation steps of the photocycle (Ludmann et al., 1998a; Der et al., 1999). When the pH was set below 1 with HCI, electric signal measured during a continuous illumination of the sample proved that the acid purple BR transports chloride ion across the membrane (D6r et al., 1991). Despite some controversial results (Moltke and Heyn, 1995), the careful study of the transient photovoltage response of the acid purple BR confirmed this conclusion (Kalaidzidis and Kaulen, 1997).

The effect of water on the electrogenic steps of the photocycle was investigated by the use of purple membranes deposited electrophoretically on a conducting glass and air-dried. The analysis of the electric signals, measured on dried oriented samples, revealed that the charge moves inside the membrane, but no net charge translocation occurs (Varo and Keszthelyi, 1983, 1985).

In wild-type BR, the excitation of intermediate M produces another photocycle, with a shortcut back to the ground state (Litvin and Balashov, 1977; Ludmann et al., 1999). Intermediate N was also proven to be photoactive (Kouyama et al., 1988; Varo and Lanyi, 1990). The excitation of intermediate K drives the photocycle directly back to the ground state (Ormos et al., 1983; Groma et al., 1995).

In this study, our purpose was to get information about the early, red-shifted K and K-like intermediates which appear in the BR photocycles. The properties of the dried sample prepared with HCI having a pH

The knowledge of the kinetic properties of dried acid purple BR (APB) is important both for basic research and application. As the properties of the K-like intermediate are changed, the use of these samples could help in the future in understanding the properties of the excited states of BR. Also, having a simple, two-state photocycle, it could be used as a data storage material.

MATERIALS AND METHODS

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Geza I. Groma, Lorand Kelemen, Agnes Kulcsar, Melinda Lakatos, and Gyorgy Varo

Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged H-6701 Hungary

Address reprint requests to Dr. Gyorgy Vdr6, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Temesvari KRT 62, H-6701 Hungary. Tel.: 36-62-432232; Fax: 36-62-433133; E-mail: varo@nucleus.szbk.u-szeged.hu.

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