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.
