Determination of the Stability of the Noncovalent Phospholipid Transfer Protein−Lipid Complex by Electrospray Time-of-Flight Mass Spectrometry

Abstract

Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein R (PI-TPR) containing either a PI, PC, or PG molecule were identified as intact complexes by nano-electrospray ionization time-of-flight mass spectrometry. The stability of these complexes in the gas phase was determined by elevating the cone voltage (cv) resulting in the appearance of the protein void of lipid. PC-TP containing a PC species carrying an sn-1 palmitoyl chain was less stable than PC-TP containing a PC species carrying an sn-1 stearoyl chain given that these complexes were dissociated for 50% at a cv of roughly 30 and 45 V, respectively. Different acyl chains on the sn-2 position did not lead to significant changes in stability of the complex. In the case of PI-TPR, the complexes containing PI and PG were dissociated for 50% at a cv of 100 V as compared to a cv of 40 V for the complex containing PC. We propose that this difference in stability is due to hydrogen bonds between the polar headgroup of PI and PG and the lipid-binding site of PI-TPR. This may explain why PI-TPR preferentially binds PI from a membrane interface.

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…This coil-helix transition upon ligand binding may to some degree explain the ''tightening'' of secondary structure we observe when SCP2 is loaded with stearoyl CoA. ''Soft'' ESI-MS has frequently been used to study biomolecular complexes in a nearly native state in the gas phase, for example protein/protein [35], enzyme/cofactor [36] and protein/lipid [25,37]. We have adapted this kind of assay to analyse SCP2/lipid.…”

“…The potential between the electrospray needle and the sample cone was generally set around 1500 V and the cone voltage (CV) was varied (between 40 and 200 V) to analyse the dissociation of the complexes. Electrospray needles were prepared as described [25]. Data were collected and analysed using MassLynx 3.5 (Waters, Elstree, UK).…”

Phosphatidylinositol transfer proteins: From closed for transport to open for exchange

“…Given the hydrogen bonding with the inositol moiety we may presume that PI is more tightly bound to the protein than PC. This was confirmed by electrospray time-of-flight mass spectrometry analysis of the PI-TPa-lipid complexes (De Brouwer et al, 2002). Both structures indicate that the sn-1 and sn-2 fatty acyl chain of either ligand occupy distinct sites with the methyl ends being close to the opening of the lipid-binding cavity which is shielded from the outside by the lipid exchange loop (helix B) and the C-terminal end (residues 261-271) as part of helix G (Fig.…”

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

“…NMR, UV circular dichroism, infrared spectroscopy and electron spin resonance). Nano-electrospray ionization time-of-flight mass spectroscopy (ESI-TOF) was developed to study the interaction of phospholipids with the phosphatidylinositol transfer protein alpha and the phosphatidylcholine transfer protein in the gas phase (1). Fluorescent modification of phosphatidylcholine (PC) was used to study its binding to the phosphatidylcholine transfer protein by fluorometric techniques (2).…”