G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality

Abstract

G protein-coupled receptor (GPCR) is incorporated into polymeric vesicles made up of diblock copolymer bilayers. Successfully incorporated GPCRs exhibit correct biased physiological orientation and respond to various ligands. After extended dehydrated storage via lyophilization and subsequent rehydration, diblock copolymer polymersomes retain their shape and incorporated GPCR retains its function.

Keywords: GPCR; bilayer vesicles; block copolymers; lyophilization; polymersomes.

Figure 1

GPCR incorporation into diblock copolymer bilayer vesicles, pGUPs. (A) Schematic of pGUP formation and protein incorporation. Films of protein, agarose, and polymer are made on a coverslip and rehydrated with a sucrose buffer solution containing BODIPY-GTPγS. pGUPs formed of diblock copolymer bilayers can be lyophilized and the GPCR retains its function (steps 2–4); for enlarged image see Supporting Information (Figure S1). (B) Confocal micrographs of pGUPs prior to lyophilization. The left micrograph shows the polymer bilayer tagged with ATTO-488-DPPE. The right micrograph shows that rhodamine antibody-tagged 5-HT_1AR is evenly distributed throughout the polymer bilayer. (C) The left image shows a pGUP sample after lyophilization. Upon rehydration, pGUPs can still be detected as shown in the right micrograph. All scale bars represent 5 µm.

Figure 2

5-HT_1AR in pGUPS display response to increasing antagonist concentration while keeping agonist concentration constant. Fluorescence unquenching due to the irreversible binding of BODIPY-GTPγS to G proteins was tracked for 12 hours for pGUPs formed with increasing amount of the antagonist (spiperone) and constant amount of agonist. Increasing the amount of antagonist in the system decreases the protein functional rate (See Table 1, for change in intensity rates). The inset shows control curves for the pGUPs that were incubated without agonist. 5-HT_1AR basal activity is captured in the pGUPs lacking agonist, spiperone only (control).

Figure 3

Functional rates of 5-HT_1AR in polymersomes (pGUPs) versus various solutions. Controls (Ctl), no agonist pGUPs, are plotted alongside agonist-exposed samples (+Ag). The percent intensity increase of the samples indicates the population of functional protein. In DI water and PBS, the 5-HT_1AR displays no fluorescence activity. In 200 mM sucrose in PBS (pH 7.4) 5-HT_1AR displays weaker fluorescence intensity increase compared to pGUPs. Furthermore there is no difference in rate between the Ctl and +Ag protein in 200 mM sucrose in PBS.