by Kobl?ek and co-workers looked at atrazine inhibition of currents obtained from PSII complexes in a glutaraldehydeCBSA matrix on a graphite electrode, reporting an IC50 of 90?nm and LOD of 2?nM (Kobl?ek et al., 2002). charge recombination in photo-oxidised reaction centres in solution showed the same selectivity of response. Titrations of reaction centre photocurrents yielded half maximal inhibitory concentrations of 208?nM and 2.1?M for terbutryn and atrazine, respectively, with limits of detection estimated at around 8?nM and 50?nM, respectively. Photocurrent attenuation provided a direct measure of herbicide concentration, with no need for model-dependent kinetic analysis of the signal used for detection or the use of prohibitively complex instrumentation, and prospects for the use of protein engineering to develop the sensitivity and selectivity of herbicide binding by the reaction centre are discussed. ((Katona et al., 2005). The overlay in Fig. 1 shows the strong similarity in backbone fold around the QB site in the two complexes and consistency in the binding conformation adopted by terbutryn (and see Supplementary Fig. 1 for a colour, stereo view of this overlay). Purple bacterial reaction centres from species such as offer a number of MRE-269 (ACT-333679) advantages as an experimental system for the development of biosensors, not the least of which is the fact that they are expressed at high levels during chemoautotrophic growth in the dark, facilitating extensive protein engineering without compromising the viability of the organism (Jones, 2009). The reaction centre is also relatively robust, one reason being that it operates at potentials that are much less oxidising than PSII, and thus it is far less prone to self-inflicted photo-oxidative damage. Open in a separate window Fig. 1 Overlay of the D, E and de -helices that form the QB pocket, and terbutryn occupying the pocket in the reaction centre (yellow (online) or light-grey (print)) and the PSII (cyan (online) or dark-grey (print)). Prepared using Protein Data Bank entries 2BNP (Katona et al., 2005) and 3PRQ (Broser et al., 2010), and PyMOL (Schr?dinger, LLC). For a stereo, colour representation see Supplementary Fig. 1. Compared to PSII there have been fewer evaluations of the use of purple bacterial reaction centres to detect herbicides. Most studies have utilised the fact that charge separation between the primary electron donor bacteriochlorophyll pair (P) and the primary (QA) and secondary (QB) acceptor quinones is blocked at the stage P+QA- in the presence of a herbicide, displacement of the QB quinone preventing formation of P+QB-. Recombination of the P+QA- radical pair occurs around 10-fold more rapidly than recombination of P+QB- (half times MRE-269 (ACT-333679) of ~100?ms and Rabbit polyclonal to AGTRAP ~1?s, respectively) and so the binding of a herbicide to the QB site will alter the kinetics of radical pair recombination after a period of illumination, or the kinetics of P photo-oxidation during a period of weak illumination, both of which can be monitored through recovery or bleaching, respectively, of a P ground state absorbance band. Several research groups have used this to study binding of herbicides or other inhibitors by purified reaction centres in solution (Jockers et al., 1993; Spyridaki et al., 2000; MRE-269 (ACT-333679) Baldini et al., 2003; Andreu et al., 2005), reconstituted into liposomes (Peters et al., 1997), or embedded in a cationic polymer (Mallardi et al., 2007; Giustini et al., 2012). Data analysis in most of these studies required the application of a mathematical model to translate changes in the kinetics of P photooxidation or subsequent recovery into the amount of herbicide binding at the QB site (e.g. see Andreu et al. (2005) and Giustini et al. (2012)). Atrazine binding has also been detected by surface plasmon resonance (SPR) through an unidentified change in the properties of reaction centres immobilised on an SPR chip (Nakamura et al., 2003). In previous work we described a simple photoelectrochemical cell based on purified reaction centres interfaced with an unfunctionalised gold electrode, and characterised the capacity of such cells to generate photocurrents under a range of conditions of illumination, applied potential and mediator concentration (den Hollander et al., 2011). Photocurrents of several hundred nA?cm?2 could be generated using reaction centres interfaced with the working and counter electrode by cytochrome and ubiquinone, respectively. In the present work we explore the use of such a photoelectrochemical cell as a biosensor by quantifying photocurrent attenuation by a range of herbicides and other quinone site inhibitors. 2.?Materials and methods 2.1. Construction and expression of His-tagged reaction centres DNA encoding a ten residue polyhistidine tag preceded by a thrombin cleavage site was designed in silico such that the resulting protein sequence was LALVPRGSSAHHHHHHHHHH. The DNA sequence was placed before the stop codon in plasmid pUCXB-1, which is.
