Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions

• A method for assembling water soluble proteins with native chlorophylls is described.
• The method is general, suitable for screening, and does not require tagged proteins.
• Chlorophylls are introduced to proteins from the oil phase of water-in-oil emulsions.
• Four recombinant WSCPs from different species were assembled with chlorophyll a.
• All WSCP complexes are homo-tetramers, binding four chlorophylls per complex.

The challenges involved in studying cofactor binding and assembly, as well as energy- and electron transfer mechanisms in the large and elaborate transmembrane protein complexes of photosynthesis and respiration have prompted considerable interest in constructing simplified model systems based on their water-soluble protein analogs. Such analogs are also promising templates and building blocks for artificial bioinspired energy conversion systems. Yet, development is limited by the challenge of introducing the essential cofactors of natural proteins that are highly water-insoluble into the water-soluble protein analogs. Here we introduce a new efficient method based on water-in-oil emulsions for overcoming this challenge. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four recombinant variants of the water-soluble chlorophyll-binding protein of Brassicaceae plants. We use the method to gain new insights into the protein–chlorophyll assembly process, and demonstrate its potential as a fast screening system for developing novel chlorophyll–protein complexes.

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