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. 2009 Feb;96(3):1016-25.
doi: 10.1016/j.bpj.2008.10.032.

Adsorption of GST-PI3Kgamma at the air-buffer interface and at substrate and nonsubstrate phospholipid monolayers

Antje Hermelink  1 , Cornelia Kirsch, Reinhard Klinger, Gerald Reiter, Gerald Brezesinski
Affiliations

Adsorption of GST-PI3Kgamma at the air-buffer interface and at substrate and nonsubstrate phospholipid monolayers

Antje Hermelink et al. Biophys J. 2009 Feb.

Abstract

The recruitment of phosphoinositide 3-kinase gamma (PI3Kgamma) to the cell membrane is a crucial requirement for the initiation of inflammation cascades by second-messenger production. In addition to identifying other regulation pathways, it has been found that PI3Kgamma is able to bind phospholipids directly. In this study, the adsorption behavior of glutathione S-transferase (GST)-PI3Kgamma to nonsubstrate model phospholipids, as well as to commercially available substrate inositol phospholipids (phosphoinositides), was investigated by use of infrared reflection-absorption spectroscopy (IRRAS). The nonsubstrate phospholipid monolayers also yielded important information about structural requirements for protein adsorption. The enzyme did not interact with condensed zwitterionic or anionic monolayers; however, it could penetrate into uncompressed fluid monolayers. Compression to values above its equilibrium pressure led to a squeezing out and desorption of the protein. Protein affinity for the monolayer surface increased considerably when the lipid had an anionic headgroup and contained an arachidonoyl fatty acyl chain in sn-2 position. Similar results on a much higher level were observed with substrate phosphoinositides. No structural response of GST-PI3Kgamma to lipid interaction was detected by IRRAS. On the other hand, protein adsorption caused a condensing effect in phosphoinositide monolayers. In addition, the protein reduced the charge density at the interface probably by shifting the pK values of the phosphate groups attached to the inositol headgroups. Because of their strongly polar headgroups, an interaction of the inositides with the water molecules of the subphase can be expected. This interaction is disturbed by protein adsorption, causing the ionization state of the phosphates to change.

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Figures

Figure 1
Figure 1
IRRA spectra of recombinant GST-PI3Kγ on Tris buffer acquired with p-polarized light at an angle of incidence of 40°. (A) GST-PI3Kγ at 5 mN/m (dashed line) and 9 mN/m (solid line). (B) Zoom-in into the amide region of the spectra presented in (A) with the same assignment of line types.
Figure 2
Figure 2
IRRA spectra of GST-PI3Kγ on Tris buffer at 9 mN/m acquired with p-polarized light at 40° and 62° (solid lines). Inserted are the corresponding simulated spectra (dotted lines) for (A) an α-helical structure lying flat at the interface, (B) a β-sheet lying flat at the air-buffer interface, and (C) a random coil structure. The spectra were calculated for an α-helix with a length of 1.5 nm and a width of 0.5 nm, and a β-sheet with a film thickness of 1 nm and the extinction coefficients of a model antiparallel β-sheet (47). The marked positions of the main bands are taken from Goormaghtigh et al.(48).
Figure 3
Figure 3
IRRA spectra of the Tris buffer surface with p-polarized light at 40° angle of incidence covered with (A) DSPC (top) and DSPA (bottom), with (solid line) and without (dashed line) GST-PI3Kγ at 5 mN/m; (B) DMPC (top) and DMPA (bottom), with GST-PI3Kγ at 5 mN/m (solid line), at 10 mN/m (dotted line), and without protein (dashed line). (C) SAPA with GST-PI3Kγ at 5 mN/m (solid line), at 30 mN/m (dotted line), and without GST-PI3Kγ (dashed line).
Figure 4
Figure 4
Influence of the headgroup structure on the GST-PI3Kγ affinity to PtdIns and PtdIns(4,5)P2 monolayers. (A) IRRA spectra of DPPtdIns (dotted line) and DPPtdIns(4,5)P2 (solid line) and (B) IRRA spectra of DOPtdIns (dotted line) and DOPtdIns(4,5)P2 (solid line), with adsorbed GST-PI3Kγ on Tris buffer at 20 mN/m and acquired with p-polarized light at 40° angle of incidence. The kinase was injected at 5 mN/m. After protein adsorption, the film was compressed to 20 mN/m.
Figure 5
Figure 5
Influence of the fatty-acid pattern on GST-PI3Kγ affinity to PtdIns(4,5)P2 monolayers. IRRA spectra of DP(16:0)- (dotted line), DO(18:1)- (dashed line), and stearoyl-arachidonoyl(18:0, 20:4)-PtdIns(4,5)P2 (solid line) with adsorbed GST-PI3Kγ on Tris buffer at 20 mN/m and acquired with p-polarized light at 40° angle of incidence. The kinase was injected at 5 mN/m. After protein adsorption, the film was compressed to 20 mN/m.
Figure 6
Figure 6
Band positions of the CH2 asymmetrical stretching modes for (A) DPPtdIns(4,5)P2 and (B) SAPtdIns(4,5)P2 on Tris buffer as a function of surface pressure Π, with (open circles, dashed lines) and without (closed circles, solid lines) GST-PI3Kγ in the subphase. The corresponding spectra were acquired with p-polarized light at 40° angle of incidence.
Figure 7
Figure 7
Phosphate band region of IRRA spectra of DPPtdIns(4,5)P2 and SAPtdIns(4,5)P2 on Tris buffer with and without GST-PI3Kγ. (A) DPPtdIns(4,5)P2 with protein at 15 mN/m (solid line) and without protein at 15 mN/m (dotted line) and at 30 mN/m (dashed line). (B) SAPtdIns(4,5)P2 with protein at 15 mN/m (solid line) and without protein at 15 mN/m (dotted line) and at 30 mN/m (dashed line). Spectra were collected with p-polarized light at 40° angle of incidence. The kinase was injected at 5 mN/m. After protein adsorption, the film was compressed to the corresponding surface pressures. The spectra (dotted-dashed line) observed with the pure protein (Fig. 1) have been added for comparison.
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