Single-Molecule Spectroscopy Selectively Probes Donor and Acceptor Chromophores in the Phycobiliprotein Allophycocyanin

Abstract

We report on single-molecule fluorescence measurements performed on the phycobiliprotein allophycocyanin (APC). Our data support the presence of a unidirectional Förster-type energy transfer process involving spectrally different chromophores, alpha84 (donor) and beta84 (acceptor), as well as of energy hopping amongst beta84 chromophores. Single-molecule fluorescence spectra recorded from individual immobilized APC proteins indicate the presence of a red-emitting chromophore with emission peaking at 660 nm, which we connect with beta84, and a species with the emission peak blue shifted at 630 nm, which we attribute to alpha84. Polarization data from single APC trimers point to the presence of three consecutive red emitters, suggesting energy hopping amongst beta84 chromophores. Based on the single-molecule fluorescence spectra and assuming that emission at the ensemble level in solution comes mainly from the acceptor chromophore, we were able to resolve the individual absorption and emission spectra of the alpha84 and beta84 chromophores in APC.

Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

“…The large heterogeneity in FPol (at low brightness) reflects the multitude of energy flow pathways on the partially bleached complexes, sampled by stochastic photobleaching. Third, some trimers display large, sudden decreases in fluorescent brightness similar to that previously observed (11,13,22) (SI Appendix, Fig. S8F).…”

“…Trimers (crosslinked to prevent dissociation, SI Appendix, Fig. S3), on the other hand, were observed to show longer trapping times and more complex stepping behavior in fluorescence brightness, similar to previous work (11,13,22).…”

“…S8 C and D). Blue-shifting of the last unbleached step was previously reported on surface-immobilized APC trimers (11,13). In addition to the major blue shift, small spectral fluctuations (∼1-5 nm) were also observed in many single-molecule traces (Fig.…”

“…Although single-pigment variants can be prepared by denaturation (8) or mutagenesis (9), the perturbation imposed by these methods might be undesirable. Single-molecule fluorescence (10)(11)(12)(13)(14)(15) can provide subcomplex information, but the common practice of immobilization might perturb the delicate integrity of these highly optimized antenna complexes. Moreover, previous studies lack sufficient spectroscopic resolution to resolve individual pigments.…”

Single-Molecule Spectroscopic Study of Enhanced Intrinsic Phycoerythrin Fluorescence on Silver Nanostructured Surfaces

“…16,17 Phycobiliproteins have also been the focus of several single-molecule spectroscopy studies and also have been used in high-speed, high-throughput, single-molecule imaging techniques for identifying in free solution biomolecules–a technique with potential for use in high-speed detection of specific disease markers. 18–21 Hence, further enhancing the fluorescence emission properties and improving the photostability of phycobiliproteins will serve to make them even more efficient labels in the analysis of biomolecules and cells. One way of doing this is via the interaction of the phycobiliproteins with silver nanostructures to achieve metal-enhanced fluorescence (MEF), which can be used to increase the photostability, brightness, and increase in radiative decay rates of fluorophores.…”