Photo-Induced Conformational Flexibility in Single Solution-Phase Peridinin-Chlorophyll-Proteins

Abstract

The peridinin-chlorophyll-protein (PCP) is an accessory light-harvesting complex found in red-tide dinoflagellates. PCP absorbs photons primarily in the blue-green spectral region via peridinin (Per) carotenoid pigments which then transfer excitations to chlorophyll (Chl) and ultimately downstream to photosystem II (PSII). Whereas the ultrafast dynamics of PCP are well-studied, much less is known about slower protein dynamics on time scales of milliseconds and seconds. Previous single-molecule studies of spectral emission and intensity have attached PCP to surfaces, but the native environment of PCP is in the lumen, meaning that a surface-attached environment could perturb its native conformations. To address this concern, we use the anti-Brownian electrokinetic (ABEL) trap to study single PCP monomers in solution for several seconds each. We measure, for the first time, simultaneous single-molecule intensity, lifetime, and spectral emission shifts for each trapped PCP monomer. The rate of reversible spectral redshifts depends linearly on irradiance over a factor of 30, indicating a light-induced mechanism which we attribute to a protein conformational change. Independent of these spectral shifts, our measurements of intensity and lifetime show reversible Chl quenching. In contrast to previous work, we show that this quenching cannot result from isolated photobleaching of Chl. These independent mechanisms arise from distinct conformational changes which maintain relatively stable fluorescence emission.

Single-Molecule Identification of Quenched and Unquenched States of LHCII

“…S2). Notably, this is in contrast to the dramatic dependence of distinct emissive states on excitation intensity observed for other photosynthetic light harvesting complexes 29,44,50 . Similarly to previously observed spectral dynamics of single LHCII complexes, 19,20 this indicates that the system appears to control the equilibrium between conformations via changes in the near environment, as opposed to conformational changes triggered directly through light intensity.…”

Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

“…Our laboratory has developed an alternative single-molecule confinement strategy, the Anti-Brownian Electrokinetic (ABEL) trap (12), which provides confinement of individual, untethered particles in free solution while monitoring photophysical parameters over the course of seconds or minutes. The ABEL trap is well-suited to investigate the time-dependent photophysical behavior of single photosynthetic antenna proteins and complexes, for example observing photodynamics (13) and unraveling pigment organization (14) in allophycocyanin, identifying conformational heterogeneity (15), and revealing photoprotective quenched states (16), among others (17,18).In this work, we have optimized the ABEL trap for simultaneous detection of brightness, polarization, emission spectrum, and fluorescence lifetime, with which we have performed a single-molecule study of the photosynthetic antenna protein C-phycocyanin (C-PC) from Spirulina platensis (19). C-PC is a water-soluble three-pigment protein found in phycobilisomes (20, 21), which are specialized antenna complexes (22) that expand spectral coverage, absorb additional energy, and provide photoprotection in cyanobacteria (23, 24).…”

“…Our laboratory has developed an alternative single-molecule confinement strategy, the Anti-Brownian Electrokinetic (ABEL) trap (12), which provides confinement of individual, untethered particles in free solution while monitoring photophysical parameters over the course of seconds or minutes. The ABEL trap is well-suited to investigate the time-dependent photophysical behavior of single photosynthetic antenna proteins and complexes, for example observing photodynamics (13) and unraveling pigment organization (14) in allophycocyanin, identifying conformational heterogeneity (15), and revealing photoprotective quenched states (16), among others (17,18).…”

Spectroscopic and transport measurements of single molecules in solution using an electrokinetic trap

“…More importantly, the ABEL trap allows synchronous fluorescent dynamics on the 10-1000ms timescale to be monitored in solution. These advances have proven to be pivotal in studying photosynthetic antenna systems at the single-molecule level 15,25,26 and could have many other applications.…”