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Photooxidative stress-inducible orange and pink water-soluble astaxanthin-binding proteins in eukaryotic microalga.

ABSTRACT: Lipid astaxanthin, a potent antioxidant known as a natural sunscreen, accumulates in eukaryotic microalgae and confers photoprotection. We previously identified a photooxidative stress-inducible water-soluble astaxanthin-binding carotenoprotein (AstaP) in a eukaryotic microalga (Coelastrella astaxanthina Ki-4) isolated from an extreme environment. The distribution in eukaryotic microalgae remains unknown. Here we identified three novel AstaP orthologs in a eukaryotic microalga, Scenedesmus sp. Oki-4N. The purified proteins, named AstaP-orange2, AstaP-pink1, and AstaP-pink2, were identified as secreted fasciclin proteins with potent ¹O₂ quenching activity in aqueous solution, which are characteristics shared with Ki-4 AstaP. Nonetheless, the absence of glycosylation in the AstaP-pinks, the presence of a glycosylphosphatidylinositol (GPI) anchor motif in AstaP-orange2, and highly acidic isoelectric points (pI = 3.6-4.7), differed significantly from that of AstaP-orange1 (pI = 10.5). These results provide unique examples on the use of water-soluble forms of astaxanthin in photosynthetic organisms as novel strategies for protecting single cells against severe photooxidative stresses.

SUBMITTER: Kawasaki S

PROVIDER: S-EPMC7477208 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Membrane proteins, particularly those that are α-helical, such as transporters and G-protein-coupled receptors (GPCRs), have significant biological relevance. However, their expression and purification pose difficulties because of their poor water solubilities, which impedes progress in this field. The QTY method, a code-based protein-engineering approach, was recently developed to produce soluble transmembrane proteins. Here, we describe a comprehensive Web server built for QTY design and its relevance for in silico analyses. Typically, the simple design model is expected to require only 2 to 4 min of computer time, and the library design model requires 2 to 5 h, depending on the target protein size and the number of transmembrane helices. Detailed protocols for using the server with both the simple design and library design modules are provided. Methods for experiments following the QTY design are also included to facilitate the implementation of this approach. The design pipeline was further evaluated using microbial transmembrane proteins and structural alignment between the designed proteins and their origins by employing AlphaFold2. The results reveal that mutants generated by the developed pipeline were highly identical to their origins in terms of three-dimensional (3D) structures. In summary, the utilization of our Web server and associated protocols will enable QTY-based protein engineering to be implemented in a convenient, fast, accurate, and rational manner. The Protein Solubilizing Server (PSS) is publicly available at http://pss.sjtu.edu.cn. IMPORTANCE Water-soluble expression and purification are of considerable importance for protein identification and characterization. However, there has been a lack of an effective method for water-soluble expression of membrane proteins, which has severely hampered their studies. Here, an enabling comprehensive Web server, PSS, was developed for designing water-soluble mutants of α-helical membrane proteins, based on QTY design, a code-based protein-engineering approach. With microbial transmembrane proteins and GPCRs as examples, we systematically evaluated the server and demonstrated its successful performance. PSS is readily available for worldwide users as a Web-based tool, rendering QTY-based protein engineering convenient, efficient, accurate, and rational.

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Photooxidative stress-inducible orange and pink water-soluble astaxanthin-binding proteins in eukaryotic microalga.

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