Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

Abstract

Oligomerization plays a critical role in shaping the light-harvesting properties of many photosynthetic pigment−protein complexes, but a detailed understanding of this process at the level of individual pigments is still lacking. To study the effects of oligomerization, we designed a single-molecule approach to probe the photophysical properties of individual pigment sites as a function of protein assembly state. Our method, based on the principles of anti-Brownian electrokinetic trapping of single fluorescent proteins, step-wise photobleaching, and multiparameter spectroscopy, allows pigment-specific spectroscopic information on single multipigment antennae to be recorded in a nonperturbative aqueous environment with unprecedented detail. We focus on the monomer-to-trimer transformation of allophycocyanin (APC), an important antenna protein in cyanobacteria. Our data reveal that the two chemically identical pigments in APC have different roles. One (α) is the functional pigment that red-shifts its spectral properties upon trimer formation, whereas the other (β) is a "protective" pigment that persistently quenches the excited state of α in the prefunctional, monomer state of the protein. These results show how subtleties in pigment organization give rise to functionally important aspects of energy transfer and photoprotection in antenna complexes. The method developed here should find immediate application in understanding the emergent properties of other natural and artificial light-harvesting systems. T he first step in photosynthesis is the capture of solar radiation by light-harvesting antenna complexes (1). In these complexes, pigments are three-dimensionally organized in a protein scaffold with specific positions, orientations, densities, and chemistry. The organizing principles (2) of these pigments are evolutionarily refined to achieve wide spectral coverage, efficient energy transport over long distances (3) and even a degree of quantum coherence (4). For example, the phycobiliproteins (5) fulfill their roles as light harvesters and energy transfer chains in cyanobacteria, by covalently binding multiple open-chain tetrapyrrole molecules (bilins), shaping the photophysical and photochemical properties of these otherwise flexible and photolabile pigments via pigment−protein interactions and forming quaternary structures that provide an extra level of pigment distance and orientation control (6).A deep understanding of the physicochemical principles of pigment organization in antenna proteins not only sheds light on the fundamentals of the light-harvesting process but can also provide guidance to the design and optimization of artificial systems (7). Critical to elucidation of these principles are the properties of the individual pigments in their native biological environment ("site properties"). However, characterization of site properties in a multipigment antenna has been difficult because the contribution of a single pigment is often obscured by signals from other pigments on the same protein. Althoug...

Orange carotenoid protein burrows into the phycobilisome to provide photoprotection

“…Nevertheless, it is also possible that a major contributor to the NPQ effect of the OCP is due to the structural modification of the core cylinder, affecting the protein environment of one or more bilin chromophores located in the area of interaction between the OCP and the PBS. This change in environment could induce interference in the native energy propagation capabilities of the affected bilin, or convert the α-or β-bilin into an energy-dissipating state, as was proposed by Wang and Moerner (42) . Further structural and spectroscopic experiments will be needed to elucidate the energyquenching mechanism.…”

“…Trimerization creates three pairs of phycocyanobilin chromophores (composed of the A84 and B84 chromophores from different monomers). These chromophore pairs may be coupled excitonically or by simple virtue of their proximity and geometry (41,42), and the centers of these pairs face into the solvent-accessible aperture. If either or both ends of hECN could approach the bilins at a distance of 5-10 Å, one might consider direct hECN-dependent NPQ.…”

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

“…To validate and refine this model, we first show that each of the experimentally observed photophysical states reasonably corresponds to one of the states predicted by this model, then numerically evaluate each state to demonstrate agreement of the predictions with our data. The simplest explanation for the diverse photophysical states observed for C-PC monomers is that they are produced through sequential photobleaching of single pigments (14), so that different combinations of pigments participate in each state, as shown in Fig. 4A.…”

“…As a check to see whether this simple photobleaching-based model appropriately describes the photophysical behavior of the C-PC monomer, we calculated the predicted B, P, τ, and emission spectrum (CM) for each state in the model, presented in SI Appendix, Table S2. Briefly, each chromophore was treated as a member of a FRET network, and the time-dependent probability of exciton residence at each participating chromophore was calculated for the cases of absorption at each participating chromophore to determine the weighted overall photophysical properties of that state (14) (Methods and SI Appendix). Fig.…”

“…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).…”