Creating a Proteoliposome Assay for Single Photosystem I Activity Assessment

Abstract

Photosystem I (PSI) is a unique photoelectronic nanomachine that produces the largest negative potential in nature, and principally sets the global enthalpy amount in all lifeforms [1]. Single molecule measurements are ideally suited to elucidate molecular level details underlying PSI activity, that remain masked in conventional ensemble PSI activity assays. These assays are usually carried out on native thylakoid membranes, containing different sizes and compositions of proteins and lipids, or on PSI solubilised in detergent, thus yielding an uninformative average activity. Here, we have employed our recently developed arrays of surface tethered single liposomes [2] on reconstituted single PSI, allowing us to monitor its activity at the single molecule level and in a massive parallel manner [3]. Liposomes constitute an ideal 3D scaffold to spatially confine single PSI in a native like environment, and can efficiently encapsulate the prefluorescent electron acceptor, resazurin [4], that upon reduction by PSI becomes highly fluorescent, thus directly yielding single PSI activity. Our studies allow us for the first time to correlate membrane characteristics (lipid composition, curvature, phase state, etc.), to regulation of PSI activity studied on the single molecule level. 1. Nelson, N. & Yocum, C. Structure and function of photosystems I and II. Plant Biology 57, (2006). 2. Hatzakis, N.S. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat Chem Biol 5, 835-841 (2009). 3. Hatzakis et al. Regulation of enzymatic activity by selection between discrete activity states probed at the single molecule level. Submitted. 4. Lohse, B., Bolinger, P. & Stamou, D. Encapsulation Efficiency Measured on Single Small Unilamellar Vesicles. J. Am. Chem. Soc 130, 14372-14373 (2008).