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Molecular cloning and biochemical characterization of the phospholipid scramblase SCRM-1 from Caenorhabditis elegans

Abstract

In this study, the SCRM-1 gene from Caenorhabditis elegans was cloned and overexpressed in E. coli to study the biochemical properties of scramblase. This is the first report showing that this scramblase from C. elegans possesses a Ca2+-dependent and head group-independent scramblase activity. The SCRM-1 of C.elegans possesses functional domains including a single EF-hand-like Ca2+ binding domain, as human scramblases do. A point mutation in the EF-hand-like Ca2+ binding motif results in loss of scramblase activity. Other biochemical assays like carbocyanine staining, Tb3+ luminescence, Tryptophan fluorescence, and CD spectroscopy strongly proved the role of the EF-hand motif for functional activity. The increase in protein size in solution upon incubating with Ca2+ shows ligand-dependent oligomerization and conformational changes.

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Acknowledgements

We acknowledge our institute and DST FIST for the facilities and University Grants Commission, Government of India for fellowship. We also thank Worm Biology lab-IITM for providing gDNA corresponding to SCRM-1. Authors thank Santosh Kumar Palanirajan for his help in editing the manuscript.

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Correspondence to Sathyanarayana N. Gummadi.

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Koyiloth, M., Gummadi, S.N. Molecular cloning and biochemical characterization of the phospholipid scramblase SCRM-1 from Caenorhabditis elegans. Eur Biophys J (2020). https://doi.org/10.1007/s00249-020-01423-2

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Keywords

  • Scramblase activity
  • EF-hand-like Ca2+ binding motif
  • Stains-All
  • Tryptophan fluorescence
  • Oligomerization