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

Abstract

Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment-protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a threepigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higherorder complexes.photosynthetic protein | C-phycocyanin | ABEL trap | single-molecule spectroscopy | photodynamics P hotosynthetic antenna structures based on pigment-protein complexes have garnered broad interest as model systems for solar energy harvesting that can directly inform development of alternative energy technologies (1). These systems transfer absorbed energy quickly and efficiently to reaction centers, and must rapidly adapt to changing irradiance to maintain photosynthetic efficiency while protecting against oxidative damage. These macroscopic behaviors of photosynthetic antenna systems are dictated by the nanoscale heterogeneous properties of the individual proteins from which they are assembled; therefore, constructing bottom-up models will be essential to fully understanding these systems. A variety of standard single-molecule techniques have been previously used to optically characterize individual antenna complexes and their substituent components, for example at low concentration (2-4) or immobilized on a surface (5-9) or in a polymer matrix (10). However, studying photosynthetic antenna proteins at the single-molecule level remains challenging (11) due to the...

Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein

“…Simultaneously, fluorescence brightness (Br), polarization (FPol), lifetime ( τ ), and emission spectrum (Em( λ ), also described by the spectral center of mass, λ CM ) are monitored for each trapped particle. The ABEL trap is well suited to characterize individual phycobiliproteins 62–64 and other photosynthetic protein complexes 65–67 because it does not require analyte immobilization, which might perturb the spectroscopic properties or energy transfer in these delicate structures (see Supplementary Note 2 for additional discussion).

Fig. 2Anti-Brownian trapping of single unquenched and quenched phycobilisomes.

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“…The ABEL trap 63 , 64 was implemented using a linearly polarized, 80 MHz pulsed-excitation laser (Coherent Mira OPO), loosely focused to a 0.6-µm beam waist in the sample plane. The beam was scanned in a knight’s tour pattern in the trapping region of a microfluidic cell over a 32-point 2 × 2 µm grid with pitch 0.4 µm.…”

“…Lifetimes were fit using a minimal single exponential model, except where otherwise noted, by a maximum likelihood method after Zander et al 71 and Brand et al 72 as previously described 62 , 64 , 73 . Briefly, each iteration of the fit model was convolved with the experimentally measured IRF, then compared with the measured lifetime decay using a Poissonian noise model to accurately determine the likelihood of that model for single-photon counting statistics.…”

Interferometric Scattering Enables Fluorescence-Free Electrokinetic Trapping of Single Nanoparticles in Free Solution

“…33 These steps must be implemented quickly enough to overcome the diffusive motion of the particle, and significant recent progress has been made toward optimizing this control loop to enable trapping of individual small organic fluorophores. 28,34 Trap implementations that utilize either intrinsic or label-based fluorescence to track emissive particles have been employed to characterize time-varying photophysical states, [35][36][37][38][39][40] molecular dynamics and kinetics, [41][42][43][44][45][46][47] and more. [48][49][50] However, the trapping duration, and therefore data collection, in anti-Brownian traps is typically limited by photobleaching or by blinking because dark particles cannot be tracked and are quickly lost.…”

Estimation of the relative contributions to the electronic energy transfer rates based on Förster theory: The case of C-phycocyanin chromophores

“…Moreover, they can also be used as natural dyes in the food industry and pharmaceuticals products [ 86 ]. From direct single-molecule measurements of C-PC in EET, by monitoring the fluorescence emission from single photosynthetic antenna proteins in a free solution over a long time and within the FRET theoretical framework, it has been found that there are transitions among many different photophysical states exhibiting distinct EET pathways among the embedded pigments [ 87 ]. These numerical findings would not be accessible by bulk studies underscoring the importance of a molecular approach.…”