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bioRxiv · 10.1101/2022.09.08.507180

A membrane-associated light harvesting model is enabled by functionalized assemblies of gene-doubled TMV proteins

Dai, J.·Wilhelm, K. B.·Bischoff, A. J.·Pereira, J. H.·Dedeo, M. T.·Garcia-Almedina, D. M.·Adams, P. D.·Groves, J. T.·Francis, M. B.
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Abstract

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light harvesting complexes embedded within phospholipid membranes. Artificial light harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Most artificial light harvesting complexes are static or in the solution phase, rather than in a two-dimensional fluid environment as in natural photosynthesis. We have developed a method for attaching a protein-based light harvesting model to a supported lipid bilayer (SLB), which provides an extended fluid membrane surface stably associated with a solid substrate. The protein model consisted of the tobacco mosaic viral capsid proteins (TMV) that were gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV were shown to break the facial symmetry of the double disk to allow for differentiation between the disk faces. Single reactive lysine and cysteine residues were incorporated into opposing surfaces of each monomer of the dTMV assemblies. This allowed for the site-selective attachment of both chromophores for light absorption and a peptide for attachment to the SLB. A cysteine modification strategy using the enzyme tyrosinase was employed for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes showed significant association with SLBs and exhibited mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light harvesting system. Significance StatementHere we have constructed a model photosynthetic membrane containing proteins, chromophores, lipids, and aqueous components, all of which can be modified in their composition. This model is based on an asymmetric disk assembly consisting of engineered tandem dimers of the tobacco mosaic viral capsid protein (dTMV). We have developed methods to achieve dye conjugation and attachment of a supported lipid bilayers (SLB) site selectively on distinct protein surfaces. These dye-labeled protein complexes exhibit mobility on the SLB, resulting in a dynamic model of light harvesting membranes using entirely synthetic components. Additionally, this unique asymmetric assembly of TMV and the facile methods for protein functionalization are expected to expand the tunability of model light harvesting systems.

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Dai, J., Wilhelm, K. B., Bischoff, A. J., Pereira, J. H., Dedeo, M. T., Garcia-Almedina, D. M., Adams, P. D., Groves, J. T., Francis, M. B.. 2022-09-10. A membrane-associated light harvesting model is enabled by functionalized assemblies of gene-doubled TMV proteins. https://doi.org/10.1101/2022.09.08.507180

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