Directed Insertion of Light-Activated Proteorhodopsin into Asymmetric Polymersomes from an ABC Block Copolymer

Abstract

Nanoscopic artificial vesicles containing functional protein transporters are fundamental for synthetic biology. Energy-providing modules, such as proton pumps, are a basis for simple nanoreactors. We report on the first insertion of a functional transmembrane protein into asymmetric polymersomes from an ABC triblock copolymer. The polymer with the composition poly­(ethylene glycol)-poly­(diisopropylaminoethyl methacrylate)-poly­(styrenesulfonate) (PEG-PDPA-PSS) was synthesized by sequential controlled radical polymerization. PEG and PSS are two distinctively different hydrophilic blocks, allowing for a specific orientation of our protein, the light-activated proton pump proteorhodopsin (PR), into the final proteopolymersome. A very interesting aspect of the PEG-PDPA-PSS triblock copolymers is that it allowed for simultaneous vesicle formation and oriented insertion of PR simply by adjusting the pH. The intrinsic positive charge of PR’s intracellular surface was enhanced by a His-tag, which aligns readily with the negative charges of the PSS on the outside of the polymersomes. The directed insertion of PR was confirmed by a light-dependent pH change of the proteopolymersome solution, indicating the intended orientation. We have hereby demonstrated the first successful oriented insertion of a proton pump into an artificial asymmetric membrane.

Polymeric Photoacids Based on Naphthols—Design Criteria, Photostability, and Light‐Mediated Release

“…The disappearance of the signala t0 .3 ppm correspondingt ot he pro- Figure 2. A) Synthesis of the photoacid-containing copolymerst hrough ad irect deprotectionofT BS protectedcopolymers, P[MEO 9 MA x -co-t"N" y ], or through ap ost-polymerization modificationoft he activated ester copolymer P(MEO 9 MA x -co-FMA y )and the respective photoacid functionalized amines (for clarity,t he RAFT end groups of the copolymers were omitted here).B)Representative 1 HNMR spectraofP [MEO 9 MA x -co-tNOeMA y ]b efore (solid black line)and afterd eprotection (solid magenta line) in CD 2 Cl 2 .C)Representative SEC elutiont races for P[MEO 9 MA x -co-tNOeMA y ]b efore (solid blackl ine) and after deprotection (solid magental ine).D )Representative 1 HNMR spectra of P[MEO 9 MA x -co-FMA y /NOeMAm y ]before(solid blackl ine) and after post-polymerization modification (solid orange line)inC D 2 Cl 2 .E)Representative 19 FNMR spectrao fP[MEO 9 MA x -co-FMA y /NOeMAm y ]before(solid blackline) and after post-polymerization modification (solido rangeline) in CD 2 Cl 2 using TFE as an internal standard. F) Representative SECelution traces for P[MEO 9 MA x -co-FMA y /NOeMAm y ]before (solid black line) and after post-polymerizationm odification (solid orange line).…”

“…[38] The reaction was conducted in DMF at 70 8Cfor two days to ensure full conversion, and 1 HNMR spectroscopy wasu sed to characterize the final functionalized copolymers ( Figure 2D). The appearance of aromatic signals corresponding to the 1-naphthol protons indicated as uccessful functionalization and the actual composition of both P[MEO 9 MA x -co-NOeMAm y ]a nd P[MEO 9 MA x -co-NAmeMAm y ]c ould be calculated and were 28 and 35 %, respectively.T of urther evaluate the extento ff unctionalization, 19 FNMR spectroscopy was carried out and ar epresentative 19 Fs pectra for P[MEO 9 MA x -co-NOeMAm y ]i ss hown in Figure 2E (the respective 19 FNMR spectra of P[MEO 9 MA x -co-NAmeMAm y ]c an be found in the SupportingI nformation, Figure S6). The characteristicf luorine signals expected for P[MEO 9 MA x -co-FMA y ]a re clearly visibleb efore and absent after the reaction.…”

“…Nuclear magnetic resonance (NMR)s pectroscopy: 1 HNMR, 13 CNMR, and 19 FNMR spectra were measured on a3 00 or 400 MHz Bruker AC spectrometer at 298 Ku sing the residual solvent resonance as an internal standard. The chemical shifts are given in ppm.…”

“…[18] In that respect, Meier and co-workers recently demonstrated the successfuli nsertion of the transmembrane protein proteorhodopsin as light-activated proton pump into asymmetric polymersomes from an ABC triblock copolymer. [19] Here, light absorp- tion by this "proteopolymersome" induced pumping of protons into the vesicles, and resulted in an increase in the extravesicular pH, turning this into the first successful example of oriented insertion of ap roton pump into an artificial asymmetric membrane. An alternative for the creation of al ocal proton gradient are photoacids, which have been known since the seminalw ork of Fçrster [20] and Weller [21] in the 1960s.…”

“…(backbone and Si(CH 3 ) 2 C(CH 3 ) 3 )), 0.3 ppm (-Si(CH 3 ) 2 C(CH 3 ) 3 ). P[MEO 9 MA x -co-PFMA y ]: 1 HNMR (400 MHz, CD 2 Cl 2 ): d = 7.88, 7.54, and 7.38 (Ar-H, CPADB), 4.3-4.0 (-(OCH 2 CH 2 -(EO) 8 -), 3.7-3.4 (-OCH 3 and -(EO) 9 -), 3.3 (-(EO) 9 -OCH 3 ), 2.2-0.7 ppm (backbone) ppm 19. FNMR (400 MHz, CD 2 Cl 2 ): d = À162.84 (2F), À158.61 (1F), À149.94 ppm (2F).…”