Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains

Abstract

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted Mr 2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.

Figure 1

Sample configuration used in direct force measurements.

Figure 2

Force vs. distance profiles were measured during initial approach between streptavidin and DSPE-EO₄₅ monolayers at different polymer grafting densities. Experiments were performed at pH 7.0 and 25°C in 10 mM NaH₂PO4 and 30 mM KNO₃. The different EO₄₅ grafting densities were 480 (•), 960 (▪), and 2,280 (○) Ų per site, which corresponded to 9.0 (•), 4.5 (▪), and 1.5 (○) mol % DSPE-EO₄₅ in the DSPE matrix.

Figure 3

The force vs. distance profile was measured between a streptavidin and a monolayer of 4.5 mol % DSPE-EO₄₅ under conditions described in the text. The best fits of the data to Derjaguin–Landau–Verwey–Overbeek theory gave −54 ± 2 mV and −41 ± 5 mV for the electrostatic potentials of the streptavidin and PEG-displaying bilayers, respectively. Similar fits were obtained using constant potential (solid line) or constant charge (dashed line) boundary conditions.

Figure 4

Hysteresis measured at the onset of adhesion between streptavidin and PEG. (A) Before the onset of attractive streptavidin-PEG interactions, there was no difference between advancing (○) and receding (•) force profiles. (B) The onset of the attraction, as signaled by hysteresis in the receding force curve (•). The surfaces finally jumped apart at the position indicated by the arrow.