Pigment-Specific Fluorescence Spectroscopy of Single Antenna Complexes in Solution

Abstract

Oligomerization plays a critical role in shaping the light-harvesting properties of many photosynthetic pigment-protein complexes (PPCs) in vivo, but a detailed understanding of this process at the level of individual pigments is still lacking. Here, aiming to study the effects of oligomerization in vitro, we designed a single-molecule approach to probe the emissive properties of individual pigment sites as a function of a PPC's different oligomerization states. Our method, based on the principles of anti-Brownian electrokinetic trapping of single fluorescent molecules, step-wise photobleaching, and multi-parameter fluorescence spectroscopy, allowed pigment-specific spectroscopic information on single PPCs to be recorded in a non-perturbative aqueous environment with unprecedented detail.In particular, we focused on the monomer-to-trimer transformation of allophycocyanin (APC), which is the core antenna complex of cyanobacteria and red algae. At the monomer level, we found that the two phycocyanobilin (PCB) pigments (α and β) show similar but non-degenerate emission properties with asymmetric FRET. Interestingly, β acts as an emissive trap that quenches the emission from α on an intact monomer and can continue to act as a non-radiative trap for α after photo-damage. In the assembled trimers, two sets of emissive properties, one resembling the β site on the monomer and the other significantly red-shifted from either of the two sites on the monomer, were revealed at the single-pigment level. These observations suggest that the bathochromatic shift that accompanies trimer assembly of APC is localized on the α pigment site, which, in the pre-assembled (monomeric) state of the protein, is shielded and protected by the β pigment.