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Bacteriorhodopsin (bR) is a trans-membrane proton pump found in the purple membrane of Halobacterium salinarum. This protein has high photochemical and photoelectric conversion efficiency and thermal stability, allowing it to withstand high temperatures, high salinity, and nutritionally-limited environments. The ability of this protein to convert light energy into chemical energy has applications that are mainly therapeutic/diagnostic and research-oriented. There is increasing demand for bacteriorhodopsin production in different fields. The present study maximized bacteriorhodopsin production using H. salinarum. The physical parameters of illumination, agitation speed, temperature, and nitrogen source were studied using a fractional factorial design to determine the optimal levels of each. The most suitable nitrogen source was determined to be peptone from meat. The optimal temperature was 39 °C, agitation speed was 150 rpm, and light intensity was 6300 lux for bR production. Under these conditions, the maximum bR yield was 196 mg/l, which is about 4.23 fold greater than those obtained with basal medium. The proposed strategies could be used for bR production using this archaeobacterium; the results are the highest reported thus far from a batch culture of H. salinarum.

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

15N T(2)' relaxation times of bacteriorhodopsin (BR) amide nitrogens were determined in the temperature range from 40 to -60 degrees C using a Hahn echo pulse sequence and proton decoupling during the echo and detection times. Using oriented membrane samples, with their bilayer normal parallel to the external magnetic field, the (15)N amide nitrogens belonging to the transmembrane helices could be selected for the analysis. The experiments were performed on purple membrane fragments (in which BR is organized in a 2D crystalline network) and on BR reconstituted into dimyristoylphosphatidylcholine at a 1:150 molar ratio (in which BR is in a freely diffusing monomeric state at 40 degrees C and in an aggregated state at 4 degrees C and below). The results are discussed in terms of helix dynamics, mosaic spread and resolution of the (15)N spectra for the various samples and experimental conditions., (Copyright 2004 John Wiley & Sons, Ltd.)

The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum., (Copyright 2003 Elsevier Science B.V.)

We present the results of two-pump and probe femtosecond experiments designed to follow the relaxation dynamics of the lowest excited state (S(1)) populated by different modes. In the first mode, a direct (S(0) --> S(1)) radiative excitation of the ground state is used. In the second mode, an indirect excitation is used where the S(1) state is populated by the use of two femtosecond laser pulses with different colors and delay times between them. The first pulse excites the S(0) --> S(1) transition whereas the second pulse excites the S(1) --> S(n) transition. The nonradiative relaxation from the S(n) state populates the lowest excited state. Our results suggest that the S(1) state relaxes faster when populated nonradiatively from the S(n) state than when pumped directly by the S(0) --> S(1) excitation. Additionally, the S(n) --> S(1) nonradiative relaxation time is found to change by varying the delay time between the two pump pulses. The observed dependence of the lowest excited state population as well as its dependence on the delay between the two pump pulses are found to fit a kinetic model in which the S(n) state populates a different surface (called S'(1)) than the one being directly excited (S(1)). The possible involvement of the A(g) type states, the J intermediate, and the conical intersection leading to the S(0) or to the isomerization product (K intermediate) are discussed in the framework of the proposed model.

Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pumping capabilities of whole cells were studied. It was found that the Delta mu (tilde)H+ of the cell had a profound influence on the kinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seen in PM preparations, we found that (1) the single-turnover time of the cycle was increased approximately 10-fold, (2) the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant for M-slow increased significantly. The level of Delta mu(tilde)H+ was dependent on respiration, ATP formation and breakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the Delta mu(tilde)H+ could be manipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levels of Delta mu(tilde)H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appeared as major components. (5) The apparent conversion of the M-fast to the O intermediate disappeared. (6) A partial reversal of an early photocycle step occurred. The photocycle of intact cells could be changed to that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. In fresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipated and proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibited by HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existing K+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were required to elicit proton extrusion.

The bacterio-opsin gene (bop) of Halobacterium halobium is located within a cluster with three other genes. Growth conditions of high light intensity and low oxygen tension induce bop gene cluster expression. To identify putative regulatory factor binding sites upstream of the bop gene, we have compared sequences upstream of the bop gene with the corresponding sequences from two other genes in the bop gene cluster. Conserved sequence motifs were observed which may mediate the effect of high light intensity and/or low oxygen tension on bop gene expression. Based on these motifs, a set of mutants was constructed which contained deletions upstream of the bop gene. These constructs were tested in a host strain where bop gene expression is independent of oxygen regulation and in another strain where it is regulated by oxygen and light. The minimal upstream sequence required for both light- and oxygen-regulated bop gene expression was determined to be 54 bp.

Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimányi, L., Váró, G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L<-->M1<-->M2 + H+ (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various pH values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L<-->M1 reaction and represents the redistribution of the proton between the Schiff base and D85. From the structure of the protein, the V49A and V49M residue replacements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed we found that they both changed the equilibrium toward the protonated Schiff base. In contrast, the residue replacements A53V and A53G were predicted to move the Schiff base in opposite directions, away from and closer to alignment with D85, respectively. The former indeed changed the equilibrium toward the protonated Schiff base and the latter toward the deprotonated Schiff base. In addition, the hydroxyl stretch band of a bound water in the L state was affected by all mutations that disfavor proton transfer to D85. We conclude that the geometry of the proton donor and acceptor in the Schiff base-D85 pair, mediated by bound water, is a determinant of the proton transfer equilibrium.

The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halopsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of detergents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.