1. Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4).
- Author
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Francis, Vincent Gerard and Gummadi, Sathyanarayana N.
- Subjects
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BIOCHEMISTRY , *PHOSPHOLIPIDS , *MEMBRANE proteins , *GENE expression , *BIOLOGICAL assay , *CARRIER proteins , *FLUORESCENCE spectroscopy - Abstract
Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affinity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp290→Ala) in hPLSCR4 decreased the Ca2+-binding affinity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ∼50% and ∼40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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