Dataset Information

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 three-pigment 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 higher-order complexes.

SUBMITTER: Squires AH

PROVIDER: S-EPMC5604012 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Squires Allison H AH Moerner W E WE

Proceedings of the National Academy of Sciences of the United States of America 20170828 37

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 ...[more]

PMID: 28847963

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Project description:Human alpha-Synuclein (alphaSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of alphaSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alphaSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy-based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant alphaSyn. The conformational heterogeneity of monomeric alphaSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and "beta-like" structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the "beta-like" structures significantly increased in different conditions promoting the aggregation of alphaSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric alphaSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric alphaSyn.

Dataset Information

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

Publications

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

Similar Datasets

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