Isoelectric Point Mobility Shift Assay for Rapid Screening of Charged and Uncharged Ligands Bound to Proteins

Abstract

SummaryThree human proteins (hTAP1, hTAP2 and hTAP3) that are related to the yeast phosphatidylinositol/phosphatidylcholine transfer protein SEC14p were recently cloned in our laboratory. These proteins contain a relatively large hydrophobic pocket, the so called CRAL-TRIO domain, which is present also in other human proteins, such as CRALBP, a-TTP and MEG2. The CRAL-TRIO domains in these proteins bind ligands such as retinaldehyde, tocopherols and polyphosphoinositides, respectively. To screen for potential hTAPs ligands, we developed a semi-quantitative isoelectric point mobility shift assay (IPMS-assay) that allows assessing the binding of potential hydrophobic ligands to proteins. Purified proteins occupied with a charged ligand migrate differently on isoelectric focusing gels when compared with free protein.Competition of bound charged ligands with uncharged ones reverses the mobility shift, so that the relative affinities of the two ligands to the protein can be estimated.

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

“…It was therefore important to check whether it can also bind αTP, which with calculated pK a values of 6.07 and 1.64, is expected to carry two negative charges with physiological condition and occurs in solution as di-sodium salt [39]. Indeed, when assayed in vitro by Isoelectric Point Mobility Shift (IPMS) assay [27], αT could compete with PI for binding to recombinant hTAP1 suggesting that the two ligands bind and depending on their concentration can exchange each other at an overlapping binding site (Figure 3A). Since 50% displacement of PI (125 µM) was observed with αT at 50 µM, the affinity of αT to the binding pocket of hTAP1 is stronger than that of PI.…”

“…Recombinant hTAP1 containing an amino-terminal Histidine tag was expressed and purified as previously described [26], [27].…”

“…The binding of αT and αTP to recombinant hTAP1 was assessed using Isoelectric Point Mobility Shift (IPMS) assay essentially as previously described [27]. In this assay, the native hTAP1 protein migrates on an isoelectric focusing polyacrylamide gel until it has a net charge of 0 (what occurs at the calculated isolectric point of recombinant hTAP1 at pH 7.9 [27]), and the mobility of hTAP1 is changed upon ligand (PI) binding, until the PI-hTAP1 complex reaches again a net charge of 0.…”

“…One group of lipids able to bind to hTAPs is related to vitamin E (α-tocopherol), encompassing the four natural tocopherol and tocotrienol analogues (α-, β-, γ-, δ-) as well as some derivatives such as α-tocopheryl quinone (αTQ) and α-tocopheryl succinate (αTS) [16], [25], [26], [27]. An intracellular tocopherol transport function of these proteins is supported by the finding that the cellular uptake of αT and αTS is increased by hTAP1 over-expression [14], [16], that the in vitro αT transport to mitochondria is augmented by hTAP1 [28], and that mitochondria-mediated apoptosis is induced by αTS in hTAP1-overexpressing mesothelioma cells and in prostate cancer cells [14], [15], [16].…”

α‐Tocopheryl phosphate – An active lipid mediator?

“…Several hydrophobic ligands are bound by the TAP proteins in vitro, such as a-, b-, g-, d-tocopherols and tocotrienols, a-tocopheryl quinone, phosphatidylcholine, phosphatidylserine, and squalene, as well as aTS, phosphatidylinositol and phosphatidylinositol-3,4,5-phosphate [47][48][49][50][56][57][58][59][60][61][62]. Using an isoelectric point mobility shift assay [59], aTP was able to compete in vitro with phosphatidylinositol for binding to recombinant human TAP1 (hTAP1) in a similar manner as aT, suggesting that aT and aTP may influence cellular events via competition for the same binding site. Since hTAP1 is the only protein so far tested for aTP binding, it is not yet clear whether hTAP2 or hTAP3 can do the same.…”