Protein-Based Model for Energy Transfer between Photosynthetic Light-Harvesting Complexes Is Constructed Using a Direct Protein-Protein Conjugation Strategy

Abstract

Photosynthetic organisms utilize dynamic and complex networks of pigments bound within light-harvesting complexes to transfer solar energy from antenna complexes to reaction centers. Understanding the principles underlying the efficiency of these energy transfer processes, and how they may be incorporated into artificial light-harvesting systems, is facilitated by the construction of easily tunable model systems. We describe a protein-based model to mimic directional energy transfer between light-harvesting complexes using a circular permutant of the tobacco mosaic virus coat protein (cpTMV), which self-assembles into a 34-monomer hollow disk. Two populations of cpTMV assemblies, one labeled with donor chromophores and another labeled with acceptor chromophores, were coupled using a direct protein-protein bioconjugation method. Using potassium ferricyanide as an oxidant, assemblies containing o-aminotyrosine were activated toward the addition of assemblies containing p-aminophenylalanine. Both of these noncanonical amino acids were introduced into the cpTMV monomers through amber codon suppression. This coupling strategy has the advantages of directly, irreversibly, and site-selectively coupling donor with acceptor protein assemblies and avoids cross-reactivity with native amino acids and undesired donor-donor or acceptor-acceptor combinations. The coupled donor-acceptor model was shown to transfer energy from an antenna disk containing donor chromophores to a downstream disk containing acceptor chromophores. This model ultimately provides a controllable and modifiable platform for understanding photosynthetic interassembly energy transfer and may lead to the design of more efficient functional light-harvesting materials.

Figure 1

Strategy for site-selective protein–protein coupling. (a) A schematic of LH1 is shown next to LH2 from R. sphaeroides. Donor chromophores are shown in green, and acceptor chromophores are shown in red. (b) A mimic for LH2-to-LH1 energy transfer is envisaged using covalently conjugated cpTMV disks labeled with fluorescent dyes. Donor pigments are shown in green, and acceptor pigments are shown in red. (c) Oxidative coupling strategies can effect the chemoselective bioconjugation of o-quinoid intermediates to anilines, N-terminal proline residues, and thiols. (d) A scheme of the oxidative coupling strategy is shown for the asymmetric conjugation of two distinct, engineered protein assemblies containing noncanonical amino acids.

Figure 2

Chemical confirmations for accessibility and modification of ncAA-containing amino acids through small-molecule couplings. (a) Conditions are shown for K₃Fe(CN)₆-mediated oxidative coupling to both cpTMV-S65-pAF (reaction A) and cpTMV-S65-3AY (reaction B). (b) Reconstructed ESI-TOF mass spectra indicated high conversion of each ncAA-containing cpTMV monomer to the expected oxidative coupling product (expected MW: 17,887 Da). (c) A cutaway view is provided, showing cpTMV monomers on opposite sides of the disk in gray, sites for protein–protein conjugation in blue, and sites for pigment attachment in red. A close-up view of a single monomer of the individual double-disk assembly is also shown.

Figure 3

Asymmetric coupling of intact cpTMV assemblies. (a) A scheme shows the covalent oxidative coupling of noncanonical amino acids on separate cpTMV disk assemblies. (b) Mass spectrometry shows both reduction of cpTMV-S65-3NY to cpTMV-S65-3AY and coupling of the monomers of the limiting protein, cpTMV-S65-3AY, at an estimated 31% yield (expected MW: 35,547 Da). (c) A size increase after oxidative coupling was verified and assemblies were isolated using SEC. Omitting the oxidant or one of the coupling partners, cpTMV-S65-pAF, prevented the size increase. (d) The increase in size and isolation of assemblies from individual disks was verified by DLS. (e) TEM images show disks coupled on their periphery through face and side views of coupled assemblies. A collection of TEM images is found in Figure S6.

Figure 4

Energy transfer between conjugated disk assemblies. (a) The donor and acceptor pair of dyes, Oregon Green 488 and Alexa Fluor 594, are shown with maleimide handles for attachment to the protein surface. (b) The absorbance spectrum of Alexa Fluor 594 and emission spectrum of Oregon Green 488, both conjugated to cpTMV assemblies, show spectral overlap. (c) A diagram of oxidatively coupled assemblies and a subset of comparison populations are shown, including cpTMV-S65-pAF disks, both sparsely (2) and completely (3) labeled with OG488, a fully labeled cpTMV-S65-pAF disk oxidatively coupled to 4-methylcatechol (4), a fully labeled cpTMV-S65-pAF disk oxidatively coupled to a cpTMV-S65-3AY disk bearing no pigments (5), a mixture of fully donor-labeled cpTMV-S65-pAF disks and fully acceptor-labeled cpTMV-S65-3AY disks which were not coupled (6), and a fully donor-labeled cpTMV-S65-pAF disk oxidatively coupled to a fully acceptor-labeled cpTMV-S65-3AY disk (7). (d) Emission spectra of coupled assemblies at an excitation wavelength of 465 nm demonstrate that emission only occurs when the donor and acceptor disks are oxidatively coupled and not when they are simply mixed. The legend indicates samples depicted in (c). (e) The fluorescence lifetime of OG488 measured at 524 nm decays more rapidly for the coupled assemblies than for comparable controls; samples are the same as those shown in (c). The inlay shows a magnified version of early timepoints for better visualization of the shortened lifetime of sample 7 in mauve.