The PD0332991 electron transfer cycle is completed by the mobile electron carrier cyt c 2 which accepts an electron from the cyt bc 1 complex, migrates to the RC and transfers an electron to reduce the oxidised primary donor (Fig. 1). The reversible binding of cyt c 2 to the reaction centre presents an attractive model system for the study membrane-extrinsic reactions but the millisecond or sub-millisecond kinetics involved places stringent demands on LDN-193189 datasheet the mapping methodology,

requiring both high temporal resolution and the ability to quantify the interaction forces. Fig. 1 Diagram of the electron transfer cycle in membranes of photosynthetic bacteria. The mobile electron carrier cyt c 2 accepts an electron from the cyt bc 1 complex and migrates to the RC and transfers an electron to reduce the oxidised primary donor In this study, we apply a newly developed

AFM-based technology for quantitative nano-mechanical imaging, PeakForce QNM (PF-QNM), to record single-molecule interactions Selleckchem Ilomastat between cyt c 2 molecules tethered to an AFM probe and RC-LH1-PufX core complexes immobilised onto a functionalised gold substrate. Intermolecular forces are quantified at the single-molecule level with nanometre spatial resolution. Kinetic data for the formation (Axelrod and Okamura 2005) and dissociation (Pogorelov et al. 2007) of the RC-cyt c 2 electron transfer complex were used to assess the performance of this new mapping technique. Results from PF-QNM are compared with those from conventional single-molecule force spectroscopy (SMFS), where imaging is not possible, but intermolecular forces can be measured. Materials and methods Protein purification RC-His12-LH1-PufX The gene encoding a RC H protein containing 12 His residues at the carboxyl terminus was created by the SLIM procedure as described (Chiu et al. 2004). The template for mutagenesis was plasmid pTZ18U::puhA, (Tehrani et al. 2003) and the four oligonucleotide primers required for this mutagenesis method were: Ft, 5′-CACCACCACCACCACCACCACCACCACCACCACCACTGATCGAGCTCTCTAGAGTCGACC-3′; Fs, 5′-CTCTAGAGTCGACCTGCAGGC-3′; Rt, 5′-AGCTCGATCAGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGGCCGCCGGCGACG-3′;

Vitamin B12 Rs, GGCCGCCGGCGACGTAGCCGCA-3′. The entire mutant gene was sequenced to confirm that only the desired change was present, and the mutant gene was subcloned as a BamHI to SacI fragment into plasmid pATP19P, (Tehrani et al. 2003) and conjugated into the ΔpuhA mutant strain of Rba. sphaeroides (Chen et al. 1998). The ΔpuhA mutant producing the 12 His-tagged RC H protein was grown semi-aerobically in 1.5 l of M22 liquid culture containing 1 mg ml−1 of tetracycline at 34 °C for 2 days in a shaker incubator (in the dark at 180 rpm). The 1.5 l culture was harvested by centrifugation (5,300 g/25 min in a Beckman JA-10 rotor at 4 °C), and the cell pellet was re-suspended in 15 ml of 10 mM HEPES pH 7.4 buffer.