Studying single plant light-harvesting complexes in near-native environments

Abstract

Photosynthesis in plants begins with light absorption by light-harvesting complexes, the main one being light-harvesting complex II (LHCII). This protein is a key component of a critical photoprotective process called non-photochemical quenching (NPQ). Single-molecule spectroscopy (SMS) has revealed that isolated LHCII complexes have complex, individual behaviour, and this behaviour can be directy related to NPQ. The physiological applicability of these findings is, however, limited by the artificial environment typically used for SMS. Applying SMS in vivo is immensely challenging, however, in part because the complexes are crowded and in constant motion. New approaches are therefore needed which enable the study of LHCII (and similar proteins) in controllable environments that mimic the native one. This thesis develops two such approaches, namely real-time feedback-driven single-particle tracking (RT-FD-SPT) and proteoliposomes, and applies them to LHCII. Different RT-FD-SPT methods were investigated using theoretical modeling, illuminating fundamental aspects of performance and aiding in the selection of an appropriate method. New data analysis code was developed for fluorescence lifetime analysis and applied to measurements on LHCII. The first measurements of photon antibunching from LHCII are reported. An RT-FD-SPT setup with unique spectroscopic measurement capabilities was constructed and used, for the first time, on light-harvesting complexes. LHCII aggregation was investigated using fluorescence correlation spectroscopy (FCS) and RT-FD-SPT. Lastly, a proteoliposome protocol was optimised which allows protein-lipid interactions to be studied at the single-molecule level.