In vitro reconstitution and biochemical characterization of human phospholipid scramblase 3: phospholipid specificity and metal ion binding studies

Abstract

Human phospholipid scramblase 3 (hPLSCR3) is a single pass transmembrane protein that plays a vital role in fat metabolism, mitochondrial function, structure, maintenance and apoptosis. The mechanism of action of scramblases remains still unknown, and the role of scramblases in phospholipid translocation is heavily debated. hPLSCR3 is the only member of scramblase family localized to mitochondria and is involved in cardiolipin translocation at the mitochondrial membrane. Direct biochemical evidence of phospholipid translocation by hPLSCR3 is yet to be reported. Functional assay in synthetic proteoliposomes upon Ca2+ and Mg2+ revealed that, apart from cardiolipin, recombinant hPLSCR3 translocates aminophospholipids such as NBD-PE and NBD-PS but not neutral phospholipids. Point mutation in hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity. Functional assay with F258V-hPLSCR3 led to ~50% loss in scramblase activity in the presence of Ca2+ and Mg2+. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes and aggregation studies. Our results revealed that Ca2+ and Mg2+ bind to hPLSCR3 and trigger conformational changes mediated by aggregation. In summary, we suggest that the metal ion-induced conformational change and the aggregation of the protein are essential for the phospholipid translocation by hPLSCR3.

Polystyrene adsorbents: rapid and efficient surrogate for dialysis in membrane protein purification

“…The NLS was slowly removed and replaced with 0.025% (w/v) Brij-35 (buffer B) from NLS supernatant by step-wise dialysis using a 10-kDa cutoff membrane. NLS was completely removed within a duration of 36 h with a buffer change of 2 L buffer B for every 12 h 21,22 . Briefly, the insoluble inclusion bodies containing the recombinant hPLSCR3 were solubilized in a buffer A containing 0.3% NLS.…”

“…Briefly, the insoluble inclusion bodies containing the recombinant hPLSCR3 were solubilized in a buffer A containing 0.3% NLS. The soluble fraction obtained after detergent solubilization was subjected to dialysis for detergent exchange (control experiment) 21 . Alternately polystyrene beads commercially called as Bio-Beads SM2 (Bio-Rad Laboratories, USA) was added to remove the detergent for further purification 23 .…”

“…The protein fraction was reconstituted into liposomes for functional assay and is termed proteoliposomes which were prepared similar to that of liposomes. To the dissolved lipid mixture, 100 µg of recombinant hPLSCR3 was added after the solubilization step and then detergent was slowly removed by Bio-Beads 19,21,22 .…”

“…The labeled liposomes and proteoliposomes collected from the previous step were resuspended in 1 ml of assay buffer and were divided into two equal fractions. The divided fractions were incubated either in the presence of 3 mM EDTA or 3 mM Ca 2+ at 37 °C for 4 h 19,21 .…”

“…The current study is focused on evaluating the efficacy of this strategy for the purification of membrane proteins as an alternate tool for conventional dialysis. Therefore to proceed further with the optimization, the recombinant 6 × His-hPLSCR3 cloned in pET28a( +) is used and the insoluble inclusion bodies are separated and solubilized with 0.3% NLS as reported earlier 19,21 . The soluble fraction was added with the Bio-Beads SM2 for detergent removal while the fraction subjected to conventional dialysis served as the control experiment ( Supplementary Fig.…”