Overview of studies of membrane protein incorporation into amphiphilic block copolymers. Most studies are done with the porn OmpF, followed by AqpZ.
Abstract
This chapter continues to further expand its focus on aquaporins (AQPs) by offering a general outline on how the AQPs block copolymers, and polymer support structures can interrelate and such connections can be comprehensively classified and defined. The first section of the overview will consider the relationship between block copolymers and AQPs. It will also examine the general membrane protein integration into block copolymers, since this can cause AQP-block copolymer complexes in vesicular (proteopolymersomes) as well as in planar forms. The majority of considerations taken into account during AQP incorporation come from the research conducted in relation to the process of incorporating other types of membrane proteins. This chapter includes an overview of the various characterization methodologies needed for the study of proteopolymersomes, as well as freeze-fracture transmission electron microscopy (FF-TEM), fluorescence correlation spectroscopy (FCS), small-angle X-ray scattering (SAXS), and stopped-flow light scattering (SFLS). The research data presented in this chapter emphasizes the fact that a successful process of membrane fabrication requires the integration of reconstituted AQPs into a suitable supporting matrix formation.
Keywords
- aquaporin proteins
- block copolymer
- matrix
- vesicular
- membrane protein
1. Assessing aquaporin proteins and block copolymer matrixes interactions
Most research performed on membrane protein inclusion has been conducted primarily with lipids as the host matrix components (original proteoliposomes publication on the subject came out in 1971) [1]. Since then, polymer-based incorporation process has received increased attention and the earliest proteopolymersomes publication emerged in 2000 [2]. The initial work stages in this research area concentrated on the inclusion of membrane-spanning proteins, such as membrane-bound ion channels (ATPases) and bacteriorhodopsin incorporation into polymethyloxazoline-polydimethylsiloxane-polymethyloxazoline (PMOXA-PDMS-PMOXA) triblock copolymer bilayers occurring in planar [3] or vesicular forms [4–6]. It is quite fascinating that the membrane proteins may be functionally incorporated into polymeric bilayers (e.g., based on PMOXA-PDMS-PMOXA) that occur up to 10 times thicker than their lipidic counterparts [7]. Moreover, researchers have observed proteopolymersomes with protein density values that drastically surpassed those of proteoliposomes [8]. Research on these phenomena has helped to establish a theoretical approach for generalized membrane protein incorporation into the amphiphilic structures. This methodology is based on the notion that the efficiency of the membrane protein incorporation process relies on its hydrophobicity potential and its coupling capacity to the host membrane is directly connected to the hydrophobic mismatch parameters. In order to reduce this mismatch dynamic, the method calls for the host membrane to be deformed in such a way that it matches the hydrophobic length parameter of the membrane protein’s transmembrane segment, in this case the hydrophobic length is 3–4 nm. A different manner of adaption would produce alternative results since the host membrane-induced membrane protein deformation is improbable due to the fact that membrane protein compressibility potential is generally one or two orders of magnitude greater than the one present in lipids [9, 10]. Researchers Srinivas and Discher argue that the application of coarse-grain simulations can ensure that the flexible hydrophobic chains would allow protein incorporation. Srinivas and Discher add that this may occur even in cases where there is a hydrophobic mismatch greater than 22% between hydrophobic interior of the chain region and membrane proteins [11, 12]. As a consequence, membrane proteins may be included with greater efficacy if the hydrophobic chains are sufficiently flexible [10]. Sine chains that are more flexible can possibly block the channel, a distinct lack of proteopolymersomes functionality can be perceived, even though the membrane protein was functionally incorporated [11]. Furthermore, elevated polydispersity may facilitate higher incorporation efficiency levels, since smaller chains can collect around the membrane protein and then offset the hydrophobic mismatch potential. Sufficiently positive incorporation data detected in the case of PMOXA-PDMS-PMOXA can thus likewise be credited to the much higher polydispersity index (PDI). Alternatively, in the setting such as natural lipid environment, the annual lipids surrounding the incorporated protein may be chosen partially due to the similarity with the lateral diffusion and protein surface [13]. The collective consequences of the hydrophobic mismatch are substantial for ATPases, ion channels [9], and co-transporter proteins. On the other hand, the effects of the mismatch are noticeably less for AQPs where they are reduced, since the protein structure itself is intrinsically more rigid [14].
Stoenescu and coworkers conducted the initial incorporation of AQPs in polymer bilayer in 2004 [15]. A research team by Stoenescu included an AQP0 originating from the mammalian eye lens directly into the polymersomes of three diverse block architectures (ABA, ABC, CBA, with A standing in for PMOXA, B for PDMS, and C for polyethylene oxide, PEO). This type of block configuration shapes how the orientation of AQP0 is being incorporated will occur. The data results obtained in this case suggest that ABA featured 50% of included AQP0 with an orientation comparable to the one occurring in liposomes, CBA included only 20%, while ABC had 80%, as shown in the antibody labeling. In all these test examples, the process of incorporation was accomplished using the addition of AQP0 content into the detergent during the polymersome configuration, as well as with the application of size exclusion chromatography (SEC) for the removal of non-incorporated protein content [15].
Kumar and coworkers were able to produce the first evidence and samples of functional AQP incorporation in 2007. Specifically, Kumar integrated bacterial AqpZ from
When it comes to the process of fabricating biomimetic membranes for a variety of applications, the original protein incorporation methodologies were from the period between 2009 and 2011 and were primarily based on the use of lipids [25, 26]. However, planar polymeric membranes have been effectively shown with the functional inclusion of gramicidin A [27]. Such research initiatives were first introduces by a Danish company called Aquaporin A/S. This company’s innovative approaches to the process of biomimetic membrane fabrication will be examined in the later sections. This researched will be supplemented with an overview of the data generated by the research groups working at the National University of Singapore (NUS) and the Singapore Membrane Technology Center (SMTC) at Nanyang Technological University (NTU).
The overview in Table 1 summarizes the critical research data on the experimental membrane protein and peptide, integration into block copolymer membranes. These data include information on a range of parameters, such as polymer chemistry and stochiometry,
Polymer | Mn | PDI | ƒ | Membrane protein | Transport Cargo | FI | mPAR | S | Incorporation method | Main functional incorporation measurement | References |
---|---|---|---|---|---|---|---|---|---|---|---|
PMOXA13-PDMS23-PMOXA13 | 3.9 | e− | X | 1:3300 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [28] | |||
PMOXA13-PDMS23-PMOXA13 | 4.7 | NA | 0.44 | Alamethicin | X | 1:590 | P | MAq | Current change | [29] | |
PMOXA13-PDMS23-PMOXA13 | 4.7 | NA | 0.44 | Hemolysin | X | 1:110,000,000 | P | MAq | Current change | [29] | |
PMOXA13-PDMS23-PMOXA13 | 4.7 | NA | 0.44 | OmpG | X | 1:33,000,000 | P | MAq | Current change | [29] | |
PMOXA20-PDMS41-PMOXA20 | 6.4 | 1.61 | 0.49 | NtAQP1 | CO2 | X | 1:360 | P | MOr | Cargo → Reaction inside vesicle → pH change | [30] |
PMOXA20-PDMS41-PMOXA20 | 6.4 | 1.61 | 0.49 | NtPIP2:1 | CO2 | X | 1:360 | P | MOr | Cargo → Reaction inside vesicle → pH change | [30] |
PMOXA20-PDMS41-PMOXA20 | 6.5 | <1.2 | 0.51 | AQP0 | H2O | ND | 10:1–1:1 | P | MAq and dialysis | [8] | |
PMOXA20-PDMS41-PMOXA20 | 6.5 | <1.2 | 0.51 | AQP0 | H2O | ND | 10:1–1:50 | V | MAq and dialysis | [8] | |
PMOXA20-PDMS41-PMOXA20 | 6.5 | <1.2 | 0.51 | AQP0 | H2O | — | 1:2.5–0 | V | MAq and dialysis | Vesicle size change | [8] |
PMOXA12-PDMS54-PMOXA12 | 6.0 | 1.01 | 0.2 | AqpZ | H2O | X | 1:100–1:1600 | V | MAq and biobeads | Vesicle size change | [31] |
PMOXA19-PDMS74-PMOXA19 | 8.7 | 1.46 | 0.23 | ||||||||
PMOXA12-PDMS54-PMOXA12 | 6.0 | 1.01 | 0.3 | AqpZ | H2O | X | 1:50–1:400 | V | MAq and biobeads | Vesicle size change | [32, 33] |
PMOXA12-PDMS54-PMOXA12 | 6.0 | 1.01 | 0.3 | Hemolysin | — | 1:83,000,000 | P | MAq | Current change | [29] | |
PMOXA20-PDMS54-PMOXA20 | 7.4 | NA | 0.42 | TsX | Nucleosides | X | 1:450 | V | MOr, SI, and SEC | Cargo → Encapsulated enzyme activity → Color change | [34] |
PMOXA8-PDMS55-PMOXA8 | 5.4 | NA | 0.22 | AqpZ | H2O | X | 1:3500 | V | PFR, biobeads, and SEC | Vesicle size change | [35] |
PMOXA12-PDMS55-PMOXA12 | 6.1 | 1.64 | 0.30 | OmpF | ELF97 | X | 1:1200 | V | MAq and SEC Cargo | Precipitation inside vesicle → Color change | [36] |
PMOXA12-PDMS55-PMOXA12 | 6.1 | 1.64 | 0.30 | OmpF | Acridine orange | X | 1:9,100,000 | V | PPFR and SEC | Cargo release → Color change | [37] |
PMOXA12-PDMS55-PMOXA12 | 6.1 | 1.64 | 0.30 | OmpF | Paraquat. Pyrocyanin | X | 1:640 | V | MAq and dialysis | No cargo → No detoxication of encapsulated enzyme → Cell death | [38, 39] |
PMOXA12-PDMS55-PMOXA12 | 6.1 | 1.64 | 0.30 | AQP0 | H2O | ND | 10:1–1:25 | P | MAq and dialysis | [8] | |
PMOXA12-PDMS55-PMOXA12 | 6.1 | 1.64 | 0.30 | AQP0 | H2O | — | 1:3–0 | V | MAq and dialysis | Vesicle size change | [8] |
PMOXA12-PDMS55-PMOXA12 | 6.1 | 1.64 | 0.30 | AqpZ | H2O | ND | 1:4 | Cr,V | MAq and biobeads | [19] | |
PMOXA7-PDMS60-PMOXA7 | 5.6 | NA | 0.19 | Gramicidin A | Monovalent cations | X | 1:81,000 | P | MOr | Current change | [27] |
PMOXA8-PDMS60-PMOXA8 | 5.8 | NA | 0.21 | ApqZ | H2O | X | 1:3800 | V | PFR, biobeads, and SEC | Vesicle size change | [35] |
PMOXA13-PDMS62-PMOXA13 | 6.8 | 1.47 | 0.29 | NADH reductase | e− | X | 1:1900 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [28] |
PMOXA15-PDMS62-PMOXA15 | 7.1 | 1.50 | 0.32 | NADH reductase | e− | X | 1:1800 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [28] |
PMOXA12-PDMS65-PMOXA12 | 6.9 | 1.67 | 0.27 | MloK1 | Potassium | X | 1:390 | P | MAq and biobeads | Current change | [40] |
PMOXA15-PDMS68-PMOXA15 | 7.6 | NA | 0.30 | LamB | Maltohexaose | X | NA | P | MAq | Current change at varying cargo concentrations | [41] |
PMOXA15-PDMS68-PMOXA15 | 7.6 | NA | 0.30 | OmpF | Actylthiocholine | X | 1:10,000 | V | PFR | Cargo → Encapsulated enzyme activity → Color change | [41] |
PMOXA15-PDMS68-PMOXA15 | 7.6 | 1.20 | 0.30 | ApqZ | H2O | X | 1:10–1:1000 | V | PFR and biobeads | Vesicle size change | [42] |
PMOXA15-PDMS68-PMOXA15 | 7.6 | 1.20 | 0.30 | Hemolysin | 1:66,000,000 | V | MAq | Current change | [29] | ||
PMOXA21-PDMS69-PMOXA21 | 8.7 | 2.00 | 0.37 | NADH reductase | e− | X | 1:1500 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [28] |
PMOXA16-PDMS72-PMOXA16 | 8.0 | 1.17 | 0.30 | OmpF | Enone | X | 1:220 | V | PPFR and dialysis | Cargo → Encapsulated enzyme activity → Color change | [43] |
PMOXA-PDMS-PMOXA | 8.8 | NA | NA | OmpF | ELF97 | X | 1:50 | V | MAq and SEC | Cargo → Precipitation inside vesicle → Color change | [44] |
PMOXA32-PDMS72-PMOXA32 | 10.7 | 1.83 | 0.47 | OmpF | 7-ADCA. PGME | X | NA | V | PFR and dialysis | Cargo → Encapsulated enzyme activity → Bacterial death | [45] |
PMOXA11-PDMS73-PMOXA11 | 7.2 | 1.70 | 0.22 | LamB | DNA | X | 1:390 | V | MOr, SI, and SEC | Fluorescence – labeled cargo | [46] |
PMOXA11-PDMS73-PMOXA11 | 7.2 | 1.70 | 0.22 | OmpF | Nucleosides | X | 1:10–1:100 | V | PPFR and SEC | Cargo → Encapsulated enzyme activity → Color change | [47] |
PMOXA11-PDMS73-PMOXA11 | 7.2 | 1.70 | 0.22 | TsX | Nucleosides | X | 1:10–1:100 | V | PPFR and SEC | Cargo → Encapsulated enzyme activity → Color change | [47] |
PMOXA11-PDMS73-PMOXA11 | 7.2 | 1.70 | 0.22 | LamB | DNA | X | NA | P | MAq | [46] | |
Lipids | |||||||||||
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | Alamethicin | Calcium | X | 1:24 | V | MAq | Cargo precipitation inside vesicle | [48, 49] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | FhuA | Sulphorhodamine B | X | 1:6,000,000 | V | MOr, SI, and SEC | Cargo → Quenching inside vesicle → Color change | [50–52] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | FhuA | TMB | X | 1:4500. 1:3,600,000 | V | MAq/ and biobeads/MOr, SI, and SEC | Cargo → Encapsulated enzyme activity → Color change | [50, 51, 53] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | FhuA | ND | 3000:1 | P | MAq | [51] | ||
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | FhuA | NAD | — | NA | V | MAq | Cargo → Encapsulated enzyme activity → Absorbance change of cargo | [52] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | FhuA | DNA | — | NA | V | MOr, SI, and SEC | Fluorescence-labeled cargo | [52] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | LamB | Sugar | X | NA | P | MAq | Current change at varying cargo concentration | [54] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | OmpF | e− | X | NA | P | MAq | Current change | [54] |
PMOXA21-PDMS73-PMOXA21 | 9.0 | 1.70 | 0.36 | OmpF | Ampicillin | X | 1:1000 | V | MOr and SEC | Cargo → Hydrolysis inside vesicle → Color change | [12, 55] |
PMOXA20-PDMS75-PMOXA20 | 9.0 | 1.46 | 0.34 | AqpZ | H2O | X | 1:25, 1:50, 1:200 | V | PFR and biobeads | Vesicle size change | [56] |
PMOXA11-PDMS76-PMOXA11 | 7.8 | 1.48 | 0.25 | BR | H+ | X | NA | V/Mc | MOr and SI | pH change | [57, 58] |
PMOXA11-PDMS76-PMOXA11 | 7.8 | 1.48 | 0.25 | BR and ATPase | H+ | X | 1:180 | V | MOr and dialysis | pH change and bioluminescence assay | [15] |
PMOXA11-PDMS76-PMOXA11 | 7.8 | 1.48 | 0.25 | BR and ATPase | H+ | X | 1:20 | V | PBR and dialysis | pH change | [59–61] |
PMOXA6-PDMS90-PMOXA6 | 9.5 | NA | 0.12 | OmpF | L-ascorbic acid, CO, Na2S2O4, ONOO− | X | 1:1300 | V | PFR, dialysis, and SEC | Cargo → Absorbance change of encapsulated protein | [62] |
PMOXA21-PDMS97-PMOXA23 | 9.0 | 1.70 | 0.30 | Hemaglutinin | X | 1:3800 | V | MAq and biobeads | MP → Fusion with fluorescence-labeled liposomes | [53] | |
PMOXA9-PDMS106-PMOXA9 | 9.4 | 1.38 | 0.14 | NADH reductase | e− | X | 1:1400 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [38] |
PMOXA13-PDMS110-PMOXA13 | 10.4 | 1.44 | 0.19 | NADH reductase | e− | X | 1:1200 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [38] |
PMOXA14-PDMS110-PMOXA14 | 10.4 | 1.36 | 0.20 | NADH reductase | e− | X | 1:1200 | V | MAq, biobeads, and SEC | Cargo → Reduction of MP → EPR signal | [38] |
PMOXA15-PDMS110-PMOXA15 | 10.7 | 1.62 | 0.21 | AqpZ | H2O | X | 1:25–1:500 | V | PFR and SEC | Vesicle size change | [7, 19] |
PMOXA15-PDMS110-PMOXA15 | 10.7 | 1.62 | 0.21 | OmpF | ND | NA | P | MAq | [53] | ||
PMOXA-PDMS-PMOXA | 20.0 | NA | FhuA | Calcein | X | 1:2,700,000 | V | MOr, SI, and SEC | Cargo release → Color change | [54] | |
PMOXA65-PDMS165-PMOXA65 | 23.3 | 1.63 | NADH reductase | e− | X | 1:550 | V | MAq, biobeads, and SEC | Cargo release → Reduction of MP → EPR signal | [28] | |
PMOXA-PDMS-PMOXA | NA | NA | NA | BR | H+ | X | NA | P | MAq | pH change | [55, 56] |
PMOXA-PDMS-PMOXA | NA | NA | NA | BR and CcO | H+ and e− | X | NA | V | MOr, SI, and SEC | Current and pH change | [56, 57] |
PMOXA-PDMS-PMOXA | NA | NA | NA | CcO | e− | X | NA | P | MOr, SI, and SEC | Current change | [55, 56] |
PMOXA-PDMS-PMOXA | NA | NA | NA | OmpF | H+ | X | NA | P | MAq | Current change | [58] |
PMOXA110-PDMS40-PEO25 | 13.4 | NA | 0.75 | AQP0 | H2O | ND | 1:200 | V | MOr, SI, and SEC | [15] | |
PMOXA45-PDMS40-PMOXA67 | 10.6 | NA | 0.68 | AQP0 | H2O | ND | 1:200 | V | MOr, SI, and SEC | [15] | |
MPEG-PVL | 6.5 | <1.2 | 0.00 | Polymyxin B | Calcein | X | 1:2 | V | MAq | Cargo release → Color change | [63] |
P2VP-PEO | NA | NA | NA | FhuA | NAD | — | NA | V | MOr, SI, and SEC | Cargo → Enzyme reaction inside vesicle → Absorbance change of cargo | [63] |
PB12-PEO10 | 1.1 | 1.09 | 0.32 | AQP0 | H2O | X | 1:5–1:250 | V | MAq and dialysis | Vesicle size change | [8] |
PB12-PEO10 | 1.1 | 1.09 | 0.32 | AQP0 | H2O | ND | 1:1.3 | Cr | MAq and dialysis | [8] | |
PB12-PEO10 | 1.1 | 1.09 | 0.32 | AQP0 | H2O | ND | 1:1–1:10 | P | MAq and dialysis | [8] | |
PB12-PEO10 | 1.1 | 1.09 | 0.3 | AqpZ | H2O | X | 1:50–1:1000 | V | MAq and dialysis | Vesicle size change | [59] |
PB12-PEO10 | 1.1 | 1.09 | 0.32 | BR | H+ | X | 1:500 | V | MAq and biobeads | pH change | [60] |
PB12-PEO10 | 1.1 | 1.09 | 0.3 | SoPIP2;1 | H2O | — | 1:200 | V | MAq and biobeads | Vesicle size change | [59] |
PB12-PEO10 | 1.1 | NA | 0.34 | Hemolysin | Calcein | X | 1:33,000 | V | MAq and dialysis | Cargo release → Color change | [61] |
PB22-PEO14 | 1.8 | 1.17 | 0.28 | AQP0 | H2O | ND | 2:1–1:300 | P | MAq and dialysis | [8] | |
PB22-PEO23 | 2.2 | 1.09 | 0.39 | AqpZ | H2O | X | 1:15–1:200 | V | MAq and dialysis | Vesicle size change | [59] |
PB22-PEO23 | 2.2 | 1.09 | 0.39 | SoPIP2;1 | H2O | — | 1:15–1:200 | V | MAq and dialysis | Vesicle size change | [59] |
PB29-PEO16 | 2.3 | 1.00 | 0.25 | AQP10 | H2O | — | 1:990 | V | PFR and SE | Vesicle size change | — |
PB35-PEO14 | 2.5 | 1.09 | 0.19 | AqpZ | H2O | — | 1:15 | V | MAq and dialysis | Vesicle size change | [59] |
PB35-PEO14 | 2.5 | 1.09 | 0.19 | SoPIP2;1 | H2O | — | 1:15 | V | MAq and dialysis | Vesicle size change | [59] |
PB43-PEO32 | 3.7 | 1.03 | 0.31 | AQP0 | H2O | X | 1:600 | V | PFR and SE | Vesicle size change | [62] |
PB46-PEO30 | 3.8 | 1.04 | 0.28 | AqpZ | H2O | — | 1:50, 1:100, 1:200 | V | MAq and dialysis | Vesicle size change | [59] |
PB46-PEO32 | 3.9 | 1.00 | 0.30 | AQP0 | H2O | — | 1:580 | V | PFR and SE | Vesicle size change | — |
PB52-PEO29 | 4.1 | <1.1 | 0.25 | Hemolysin | e− | X | NA | P | MAq | Current change | [64] |
PB52-PEO29 | 4.1 | <1.1 | 0.25 | Polymyxin B | X | NA | P | MAq | Current change | [65] | |
PB52-PEO29-LA | 4.1 | <1.1 | 0.25 | Hemolysin | e− | X | NA | P | MAq | Current change | [64] |
PB52-PEO29-LA | 4.1 | <1.1 | 0.25 | Polymyxin B | X | NA | P | MAq | Current change | [65] | |
PB92-PEO78 | 8.4 | 1.08 | 0.34 | AQP0 | H2O | — | 1:270 | V | PFR and SE | Vesicle size change | — |
PB125-PEO80 | 8.9 | <1.1 | 0.28 | Alamethicin | Calcein | — | 1:2–1:8 | V | MAq | Cargo release → Color change | [66] |
PHEMA25-PBMA25-PHEMA25 | 14.3 | 1.30 | 0.83 | AqpZ | — | NA | P | MAq and biobeads | Current change | [67] | |
PHEMA25-PBMA25-PHEMA25 | 14.3 | 1.30 | 0.83 | Hemolysin | X | NA | P | MAq | Current change | [67] | |
PHEMA25-PBMA25-PHEMA25 | 14.3 | 1.30 | 0.83 | OmpF | — | 1:70 | P | MAq and biobeads | Current change | [67] | |
PEE37-PEO40 | 3.9 | <1.1 | 0.39 | Alamethicin | Calcein | X | 1:2–1:8 | V | MAq | Cargo release → Color change | [66] |
PPO34-PGM14 | 6.5 | 1.30 | 0.66 | Strepatividin-BSA | ND | 1:5, 1:15, 1:50 | V | PPFR | [68] | ||
PI93-PEO87 | 10.2 | 1.00 | 0.31 | FhuA | TMB | X | 1:6700, 1:5,300,000 | V | MOr, SI, and SEC | Cargo → Encapsulated enzyme activity → Color change | [63] |
PEO136-PIB18-PEO136 | 8.0 | 1.86 | 0.90 | Cecropin A | Calcein | X | 1:30 | V | MAq and SEC | Cargo release → Color change | [69] |
P4MVP21-PS26-P4MVP21 | 13.1 | NA | 0.80 | PR | X | 1:10 | V | MAq and precipitation | Absorbance change in membrane protein | [70] | |
P4MVP21-PS38-P4MVP21 | 14.3 | 1.19 | 0.74 | PR | X | 1:10 | V | MAq and precipitation | Absorbance change in membrane protein | [70] | |
P4MVP29-PS42-P4MVP29 | 18.7 | NA | 0.78 | PR | X | 1:10 | V | MAq and precipitation | Absorbance change in membrane protein | [70] | |
P4MVP22-PB28-P4MVP22 | 15.0 | NA | 0.92 | PR | ND | 1:10 | V | MAq and precipitation | [71] | ||
P4MVP22-PB28-P4MVP22 | 15.0 | NA | 0.92 | RC | e− | X | 1:25 | V | MAq and precipitation | Cargo |
[72] |
P4VP22-PB28-P4VP22 | 7.1 | NA | 0.82 | PR | ND | 1:10 | V | MAq and precipitation | [71] | ||
P4MVP29-PB56-P4MVP29 | 17.4 | 1.08 | 0.81 | RC | e− | X | 1:25 | V | MAq and precipitation | Cargo |
[72] |
P4MVP18-PB93-P4MVP18 | 13.9 | 1.06 | 0.62 | PR | ND | 1:10 | V | MAq and precipitation | [71] |
An overview in Figure 3 outlines data on membrane protein integration into polymers in cases where
2. Evaluation of AQP incorporation characterization methodologies
The process of identifying examples of functional AQPs incorporation may strike as potentially quite challenging, since the permeating solute is composed of neutral water molecules. The protein-mediated type of transport when it comes to neutral molecules, and specifically at the single protein levels, is consistently more difficult to assess than the transport parameters of charged molecules, such as ions or protons or in cases of specific chemical reactions, including the ATPase enzyme activity. While the deuterated water labeling was suggested as a measurement method using the Raman spectroscopy [84], researchers are concerned that these approaches to measurement can be further complicated because the water transport rate value in the AQP channel varies for deuterated water molecules when compared to the normalized water molecule rate [85]. SFLS is a common methodology used for calculating the functional integration. SFLS method relies on a dynamic where the proteopolymersomes are vigorously combined with an osmotically active agent (NaCl or sucrose) within a specifically defined amount of volume. If a hyperosmotic shock occurs, the proteopolymersomes will become smaller in size and this in turn will facilitate light scattering to increase. Once the content of incorporated AQPs is augmented, the overall shrinking rate will likewise begin to increase. Nonetheless, the SFLS approach is substantially influenced by the quality, or size distribution potential, of the polymersomes, the concentration of the osmolytes, and the AQP concentration within the polymersomes [35]. In theory, a visually based measurement can be accomplished using the freeze-fracture transmission electron microscopy (FF-TEM), however, the FF-TEM will not be able to provide sufficient data on the functional aspects. During the FF-TEM assessment, the proteopolymersomes are caught in their original shape with the aid of the quick-freezing process. After the proteopolymersomes are collected, the frozen sample is then fractured in such as manner that the fracture plane is located alongside the proteopolymersome bilayer, since this section is the most vulnerable point in the entire system. The experimental samples with integrated AQPs, or the cavities in which the AQPs were inserted within the bilayer, are subsequently exposed to the carbon/metal coating process. The replica formed during this procedure is then detached from the thawed out sample. As a result, the AQPs and cavities can be viewed and examined on the formed replica in the shape of separate spots on the proteopolymersomes content.
Alternative methodology available is the fluorescence correlation spectroscopy (FCS) process of the fluorescently labeled AQP. The FCS is based on the time-dependent fluctuations of fluorescence intensities within a defined microscopic space, otherwise known as the confocal volume, which are carefully observed and then exposed to autocorrelation function process. The specific number of particles within the confocal volume at given time interval can be calculated, however it depends on the diffusion times of particles spreading through the confocal volume. After the proper proteopolymersomes or proteoliposomes monitoring process, they are solubilized to micelles and monitored once more so as to calculate the proteins-per-vesicle-ratio, or the primary number of membrane proteins integrated into the bilayer of a single vesicle. Within this experimental scenario, it is presupposed that the micelles include only one AQP, and as a result the micelle-per-vesicle ratio is equivalent to the proteins-per-vesicle-ratio value. Additional information on the methodology is provided in Ref. [83]. On the other hand, it is possible to calculate the proteins-per-vesicle-ratio using a correlation between the proteopolymersome solution data and the AQP stock solution parameters.
In both of the outlined methodologies, the overall correlations of data have certain benefits as well as challenges, and these are outlined in greater detail in the FCS subsection. Small-angle X-ray scattering (SAXS) capacity to characterize the biological materials makes it an adaptive toolkit when it comes to particle structure. For instance, it can supply structural data about particles in a solution on a long-scale from 1 to 100 nm, and where the collected data is shown using scattering intensity values as a function of the magnitude parameter of the scattering vector
The upcoming section is an overview of SFLS, FF-TEM, FCS, and SAXS analyses featuring a variety of diblock copolymers containing optimal
2.1. Stopped-flow light scattering
An illustration of the SFLS and its analysis, the information about PB45-PEO14 and PB33-PEO18 diblock copolymer proteo- and polymersomes, specifically with or without AqpZ, is outlined in Figure 4. In the case of PB33-PEO18, the rate constant value related to the augmentation of the light scattering intensity was somewhat greater when using AqpZ, while for PB45-PEO14, the rate it was even smaller. Such a dynamic demonstrates one of the major issues that exist with respect to the SFLS application. The distinct lack of substantial response to the alterations in extra vesicular osmolarity can be caused by the growth in the bilayer bending modulus caused by the existence of ApqZ, either blocked or non-functional. Analogous concerns have been noted in previous experimental runs using AqpZ as well as SoPIP2, and where only polymers of the smallest size (PB12-PEO10 and PB22-PEO23) showed a considerable change in SFLS between proteo- and polymersomes (the results are not shown). Additional explanation for the analogous SFLS signal could be found in the blockage of AqpZ channels with PEO chains. In this type of blockage, the AqpZ are situated directly in the bilayer and act as an impermeable hydrophobic blockage. Research conducted by Kumar et al. [8] indicates that this blockage dynamic is caused by the water permeation that is actively blocked by the sections corresponding to the integrated AqpZ, and where the proteopolymersomes’ lower permeability values can be expected, if compared to the values occurring when using polymersomes. Alternatively, the incorporated AqpZ may continue being fully functional, while the polymer matrix remains resistant to various changes in volume parameters. These phenomena effectually undermine the idea that SFLS is not a stand-alone type of technique.
2.2. Freeze-fracture transmission electron microscopy
The summary of research data on FF-TEM for PB45-PEO14 proteopolymersomes is shown in Figure 5. Specifically, proteopolymersomes featuring an mPAR of 1:100 were created with the help of film rehydration (FR) approach, where they are frozen and then fractured in a Leica MED20 station. In the next step, the two planchets of the frozen sample are carefully separated, causing an intentional fracture that simulates something like a “crack” rather than a “cut” shape, which in turn lowers the smearing effects from the conventional FF procedures (for additional details refer to relevant supporting information). It is likely that because of the collapsed PB chains, all proteo- and polymersomes featured a distinguishable surface similar to a raspberry. In fact, the “typically” present spots that studies on proteoliposomes are usually associating with AQP [86] were not found. Figure 5 outlines the bubble-like spots and their equal distribution among the polymersomes (Figure 5a–d) and proteopolymersomes (b, c, e, f). These bubble resembling spots may be PB chain accumulations (Figure 5a–c), or alternatively, phenomena produced by inferior fracturing (d–f) quality. Similar behavior was seen in proteo- and polymersomes in other PB-PEO polymers at different
2.3. Fluorescence correlation spectroscopy
FF-TEM and SFLS can result in several issues when applied as tools for potential evaluation of protein incorporation into polymersomes. As an alternative, FCS was examined as a possible method for collecting relevant quantitative data about the AQP incorporation process. The turn to FCS as a method was motivated by a recent Erbakan
Research conducted suggests that the example correlation relies on the sole constituent of the system. For instance, when it comes to sensitive fluorophores, it is more important to comparatively analyze the AQP vesicles with the AQP micelles so as limit the potential impact on the fluorophore environment. With polymers, including the protein matrix, it is more advantageous to relate the AQP-fluorophore stock solution since the polymeric AQP micelles are capable of aggregating more easily. The difficulty caused by the correlation of AQP vesicles with AQP-fluorophore stock is that the resulting concentration value of AQP remains undetermined, and this seriously obscures the potential correlation with analogous AQP concentrations. Based on the species amount of pure AQP10-GFP in the confocal volume stock and the quantity from the proteopolymersome solution (Figure 6a), the proteins-per-vesicle-ratio was calculated as 2.87. The data obtained during these experimental runs show that FCS can in fact be applied as a dependable method for calculating AQPs in proteopolymersomes. This in turn invites a new opportunity for conducting a methodical research study where
2.4. Small-angle X-ray scattering
Figure 7 showcases the scattering curves for FR prepared proteo- and polymersomes for PB33-PEO18 and PB45-PEO14. These examples went through a process of extruding and centrifuging before the actual measurements were taken. The typical linear slope was detected at low
In cases focusing on the theoretical scattering in assorted straightforward geometrical objects, including spheres, ellipsoids, and cylinders of different contrast, the scattering values can be carefully calculated with relative ease. In fact, this data can be collected so as to form simplified models of the particles being studied. In this research model, the data were examined with the aid on a vesicle model based on three concentric spherical shells featuring interchanging contrast values and matched to shells of PEO, PB, and PEO. The thickness values varied in each individual shell so as to ensure the data fit and accuracy through the application of the least squares fitting routine. Exceptionally good fit correlations were found for the PB45-PEO14-system, further suggesting that the research data were in excellent agreement with the theoretical presupposition that diblock copolymers could form spherical vesicle structures. The correlated fits were shown to be especially sensitive to fluctuations in the factors that affect the central hydrophobic bilayer thickness founded using the PB-groups. These values were found to be in the ranges of 9.10 ± 0.1 and 8.94 ± 0.07 nm in the cases where AqpZ was either present or absent, respectively. With regards to the overall vesicle diameter value, the model suggests that it is greater than 60 nm, a result that aligns well with the initial analysis of the system. The collected data reflects that the fit parameters defining bilayer vesicles are created and that the inclusion of AqpZ incites very slight changes in the vesicles’ structure.
In the case of the PB33-PEO18 proteopolymersomes, sufficiently suitable fits were obtained with the vesicle model based on a hydrophobic bilayer with the thickness values of 7.66 ± 0.05 nm. On the other hand, for experimental runs with polymersomes, there were no adequate fit options for the data that would ensure realistic physical parameters. In fact, for the data to fit the experimental approach needed to assume that the vesicles could coexist with a population of block copolymer micelles. The combined model fit parameters suggested that 76 wt% of the population was comprised of proteopolymersomes, and that 24 wt% were micelles with hydrophobic cores of diameter 11.7 ± 0.3 nm, thus showing a relatively good fit with the data overall. The Figure 7 insert outlines the separate micelle and vesicle contributions.
To sum up, the SAXS inquiry exposes that in the case of for PB45-PEO14 the vesicles are created both with, as well as without, the AQP, where the AQP incorporation leads to a small variance in the average hydrophobic vesicle wall thickness value, and can imply a polymer puckering and dimpling near the integrated AQPs. For the PB33-PEO18 experimental runs, some micelle creation was seen, but this formation becomes lower once the AQP is successfully integrated. To sum up, this chapter examines the research that explored the characterization methods used for the functional integration of AQPs in PB-PEO diblock copolymers. The research results obtained suggest that both FF-TEM and SFLS are in theory effective methods, however, when it comes to polymer systems the critical analysis of findings can provide ambiguous data that makes it problematic for applications. Alternatively, SAXS and FCS have been evaluated as capable of producing relevant information, with SAXS relying on access to large-scale facilities that can sustain synchrotron radiation resources.
References
- 1.
Kagawa Y, Racker E. Partial resolution of the enzymes catalyzing oxidative phosphorylation. The Journal of Biological Chemistry. 1971; 246 :5477-5487 - 2.
Nardin C, Thoeni S, Widmer J, Winterhalter M, Meier W. Nanoreactors based on (polymerized) ABA-triblock copolymer vesicles. Chemical Communications. 2000:1433-1434 - 3.
Zhang X, Tanner P, Graff A, Palivan CG, Meier W. Mimicking the cell membrane with block copolymer membranes. Journal of Polymer Science, Part A: Polymer Chemistry. 2012; 50 :2293-2318 - 4.
Tanner P, Baumann P, Enea R, Onaca O, Palivan C, Meier W. Polymeric vesicles: From drug carriers to nanoreactors and artificial organelles. Accounts of Chemical Research. 2011; 44 :1039-1049 - 5.
Choi HJ, Montemagno C. Artificial organelle: ATP synthesis from cellular mimetic polymersomes. Nano Letters. 2005; 5 :2538-2542 - 6.
Vriezema DM, Garcia PML, Sancho Oltra N, Hatzakis NS, Kuiper SM, Nolte RJM, Rowan AE, van Hest JCM. Positional assembly of enzymes in polymersome nanoreactors for cascade reactions. Angewandte Chemie, International Edition. 2007; 46 :7378-7382 - 7.
Kumar M, Grzelakowski M, Zilles J, Meier WP. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein aquaporin Z. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104 :20719-20724 - 8.
Kumar M, Habel J, Shen YX, Meier W, Walz T. High-density reconstitution of functional water channels into vesicular and planar block copolymer membranes. Journal of the American Chemical Society. 2012; 134 :18631-18637 - 9.
Andersen O. Bilayer thickness and membrane protein function: An energetic perspective. Annual Review of Biophysics and Biomolecular Structure. 2007; 36 :107-130 - 10.
Pata V, Dan N. The effect of chain length on protein solubilization in polymer-based vesicles (polymersomes). Biophysical Journal. 2003; 85 :2111-2118 - 11.
Srinivas G, Discher D, Klein M. Key roles for chain flexibility in block copolymer membranes that contain pores or make tubes. Nano Letters. 2005; 5 :2343-2349 - 12.
Discher DE, Ortiz V, Srinivas G, Klein ML, Kim Y, Christian D, Cai S, Photos P, Ahmed F. Emerging applications of polymersomes in delivery: From molecular dynamics to shrinkage of tumors. Progress in Polymer Science. 2007; 32 :838-857 - 13.
Aponte-Santamaría C, Briones R, Schenk A, Walz T, de Groot B. Molecular driving forces defining lipid positions around aquaporin-0. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109 :9887-9892 - 14.
Hansen JS, Vararattanavech A, Plasencia I, Greisen PJ, Bomholt J, Torres J, Emnéus J, Hélix-Nielsen C. Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: A comparative study of spinach SoPIP2;1 and E. coli AqpZ. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2011;1808 :2600-2607 - 15.
Stoenescu R, Graff A, Meier W. Asymmetric ABC-triblock copolymer membranes induce a directed insertion of membrane proteins. Macromolecular Bioscience. 2004; 4 :930-935 - 16.
Hite RK, Li Z, Walz T. Principles of membrane protein interactions with annular lipids deduced from aquaporin-0 2D crystals. The EMBO Journal. 2010; 29 :1652-1658 - 17.
Nehring R, Palivan CG, Casse O, Tanner P, Tüxen J, Meier W. Amphiphilic diblock copolymers for molecular recognition: Metal-nitrilotriacetic acid functionalized vesicles. Langmuir. 2009; 25 :1122-1130 - 18.
Kelly D, Abeyrathne P, Dukovski D. The affinity grid: A pre-fabricated EM grid for monolayer purification. Journal of Molecular Biology. 2008; 382 :423-433 - 19.
Kumar M. Biomimetic membranes as new materials for applications in environmental engineering and biology. Ph.D. Thesis. Champaign, IL, USA: University Illinois; 2010 - 20.
Kumar M, Meier W. Highly Permeable Polymeric Membranes. Patent WO 2009/078174; 18-06-2009 - 21.
Gonen T, Sliz P, Kistler J, Cheng Y, Walz T. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature. 2004; 429 :193-197 - 22.
Chandy G, Zampighi G, Kreman M, Hall J. Comparison of the water transporting properties of MIP and AQP1. The Journal of Membrane Biology. 1997; 159 :29-39 - 23.
Kumar M, Walz T. High Density Membrane Protein Membranes. Patent WO 2014/028923; 20-02-2014 - 24.
Habel J. Structural and functional characterization of aquaporin 0 incorporated in block copolymers and their resulting aggregate morphologies. Master Thesis. Basel, Switzerland: Universität Basel; 2011 - 25.
Hélix-Nielsen C. Biomimetic membranes for sensor and separation applications. Analytical and Bioanalytical Chemistry. 2009; 395 :697-718 - 26.
Pszon-Bartosz K, Hansen JS, Stibius KB, Groth JS, Emnéus J, Geschke O, Hélix-Nielsen C. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter. Biochemical and Biophysical Research Communications. 2011; 406 :96-100 - 27.
González-Pérez A, Stibius K, Vissing T. Biomimetic triblock copolymer membrane arrays: A stable template for functional membrane proteins. Langmuir. 2009; 25 :10447-10450 - 28.
Graff A. Amphiphilic copolymer membranes promote NADH: Ubiquinone oxidoreductase activity: Towards an electron-transfer nanodevice. Macromolecular Chemistry and Physics. 2010; 211 :229-238 - 29.
Wong D, Jeon TJ, Schmidt J. Single molecule measurements of channel proteins incorporated into biomimetic polymer membranes. Nanotechnology. 2006; 17 :3710-3717 - 30.
Uehlein N, Otto B, Eilingsfeld A, Itel F, Meier W, Kaldenhoff R. Gas-tight triblock-copolymer membranes are converted to CO2 permeable by insertion of plant aquaporins. Scientific Reports. 2012; 2 :1-4 - 31.
Wang H, Chung TS, Tong YW. Study on water transport through a mechanically robust aquaporin Z biomimetic membrane. Journal of Membrane Science. 2013; 445 :47-52 - 32.
Wang H, Chung TS, Tong YW, Jeyaseelan K, Armugam A, Chen Z, Hong M, Meier W. Highly permeable and selective pore-spanning biomimetic membrane embedded with aquaporin Z. Small. 2012; 8 :1185-1190 - 33.
Zhong PS, Chung TS, Jeyaseelan K, Armugam A. Aquaporin-embedded biomimetic membranes for nanofiltration. Journal of Membrane Science. 2012; 407-408 :27-33 - 34.
De Vocht C, Ranquin A, Willaert R, Van Ginderachter JA, Vanhaecke T, Rogiers V, Versées W, Van Gelder P, Steyaert J. Assessment of stability, toxicity and immunogenicity of new polymeric nanoreactors for use in enzyme replacement therapy of MNGIE. Journal of Controlled Release. 2009; 137 :246-254 - 35.
Grzelakowski M, Cherenet MF, Shen YX, Kumar M. A framework for accurate evaluation of the promise of aquaporin based biomimetic membranes. Journal of Membrane Science. 2015:1-32 - 36.
Grzelakowski M, Onaca O, Rigler P, Kumar M, Meier W. Immobilized protein-polymer nanoreactors. Small. 2009; 5 :2545-2548 - 37.
Ihle S, Onaca O, Rigler P, Hauer B, Rodríguez-Ropero F, Fioroni M, Schwaneberg U. Nanocompartments with a pH release system based on an engineered OmpF channel protein. Soft Matter. 2011; 7 :532-539 - 38.
Tanner P, Balasubramanian V, Palivan CG. Aiding nature’s organelles: Artificial peroxisomes play their role. Nano Letters. 2013; 13 :2875-2883 - 39.
Tanner P, Onaca O, Balasubramanian V, Meier W, Palivan CG. Enzymatic cascade reactions inside polymeric nanocontainers: A means to combat oxidative stress. Chemistry - A European Journal. 2011; 17 :4552-4560 - 40.
Kowal JŁ, Kowal JK, Wu D, Stahlberg H, Palivan CG, Meier WP. Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports. Biomaterials. 2014; 35 :7286-7294 - 41.
Winterhalter M, Hilty C, Bezrukov SM, Nardin C, Meier W, Fournier D. Controlling membrane permeability with bacterial porins: Application to encapsulated enzymes. Talanta. 2001; 55 :965-971 - 42.
Xie W, He F, Wang B, Chung TS, Jeyaseelan K, Armugam A, Tong YW. An aquaporinbased vesicle-embedded polymeric membrane for low energy water filtration. Journal of Materials Chemistry A. 2013; 1 :7592-7600 - 43.
Heinisch T, Langowska K, Tanner P, Reymond JL, Meier W, Palivan C, Ward TR. Fluorescence-based assay for the optimization of the activity of artificial transfer hydrogenase within a biocompatible compartment. ChemCatChem. 2013; 5 :720-723 - 44.
Broz P, Driamov S, Ziegler J, Ben-Haim N, Marsch S, Meier W, Hunziker P. Toward intelligent nanosize bioreactors: A pH-switchable, channel-equipped, functional polymer nanocontainer. Nano Letters. 2006; 6 :2349-2353 - 45.
Langowska K, Palivan CG, Meier W. Polymer nanoreactors shown to produce and release antibiotics locally. Chemical Communications. 2013; 49 :128-130 - 46.
Graff A, Sauer M, Meier W. Virus-assisted loading of polymer nanocontainer. Proceedings of the National Academy of Sciences of the United States of America. 2002; 99 :5064-5068 - 47.
Ranquin A, Versées W, Meier W, Steyaert J, Van Gelder P. Therapeutic nanoreactors: Combining chemistry and biology in a novel triblock copolymer drug delivery system. Nano Letters. 2005; 5 :2220-2224 - 48.
Lee H, Ho D, Kuo K, Montemagno CD. Vectorial insertion of bacteriorhodopsin for directed orientation assays in various polymeric biomembranes. Polymer. 2006; 47 :2935-2941 - 49.
Choi HJ, Germain J. Effects of different reconstitution procedures on membrane protein activities in proteopolymersomes. Nanotechnology. 2006; 17 :1825-1830 - 50.
Choi HJ, Montemagno CD. Biosynthesis within a bubble architecture. Nanotechnology. 2006; 17 :2198-2202 - 51.
Choi HJ, Montemagno C. Light-driven hybrid bioreactor based on protein-incorporated polymer vesicles. IEEE Transactions on Nanotechnology. 2007; 6 :171-176 - 52.
Dobrunz D, Toma AC, Tanner P, Pfohl T, Palivan CG. Polymer Nanoreactors with dual functionality: Simultaneous detoxification of peroxynitrite and oxygen transport. Langmuir. 2012; 28 :15889-15899 - 53.
Thoma J, Belegrinou S, Rossbach P, Grzelakowski M, Kita-Tokarczyk K, Meier W. Membrane protein distribution in composite polymer-lipid thin films. Chemical Communications. 2012; 48 :8811-8813 - 54.
Onaca O, Sarkar P, Roccatano D, Friedrich T, Hauer B, Grzelakowski M, Güven A, Fioroni M, Schwaneberg U. Functionalized nanocompartments (synthosomes) with a reductiontriggered release system. Angewandte Chemie, International Edition. 2008; 47 :7029-7031 - 55.
Ho D, Chu B, Lee H, Montemagno C. Protein-driven energy transduction across polymeric biomembranes. Nanotechnology. 2004. DOI: 10.1088/0957-4484/15/8/038 - 56.
Ho D, Chu B, Lee H, Brooks EK, Kuo K, Montemagno CD. Fabrication of biomolecule–copolymer hybrid nanovesicles as energy conversion systems. Nanotechnology. 2005; 16 :3120-3132 - 57.
Xi JZ, Ho D, Chu B, Montemagno CD. Lessons learned from engineering biologically active hybrid nano/micro devices. Advanced Functional Materials. 2005; 15 :1233-1240 - 58.
Ho D, Chu B, Schmidt JJ, Brooks EK, Montemagno CD. Hybrid protein-polymer biomimetic membranes. IEEE Transactions on Nanotechnology. 2004; 3 :256-263 - 59.
Habel J. Functional and Chemical Characterization of Vesicular Diblock Copolymer Bilayers with Aquaporin Included. Technical Report, Aquaporin A/S, Copenhagen, Denmark; 2011 - 60.
Espina M. Barrier properties of biomimetic membranes. Master Thesis. Lyngby, Denmark: Danish Technical University (DTU) Kgs; 2012 - 61.
Nallani M, Andreasson-Ochsner M, Tan CWD, Sinner EK, Wisantoso Y, Geifman-Shochat S, Hunziker W. Proteopolymersomes: In vitro production of a membrane protein in polymersome membranes. Biointerfaces. 2011;6 :153-157 - 62.
Bomholt J. Human Aquaporins—From in vivo detection to industrial scale production. Ph.D. Thesis. Copenhagen, Denmark: University Copenhagen; 2014 - 63.
Onaca O. Functionalized polymer vesicles and interactions with Polymyxin B and derivates. Ph.D. Thesis. Bremen, Germany: Universität Bremen; 2007 - 64.
Zhang X, Fu W, Palivan CG, Meier W. Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane. Scientific Reports. 2013; 3 . DOI: 10.1038/srep02196 - 65.
Dorn J, Belegrinou S, Kreiter M, Sinner EK, Meier W. Planar block copolymer membranes by vesicle spreading. Macromolecular Bioscience. 2011; 11 :514-525 - 66.
Vijayan K, Discher DE, Lal J, Janmey P, Goulian M. Interactions of membrane-active peptides with thick, neutral, nonzwitterionic bilayers. The Journal of Physical Chemistry. B. 2005; 109 :14356-14364 - 67.
Toughrai S. Functional surfaces through biomimetic block copolymer membranes. Ph.D. Thesis. Basel, Switzerland: Universität Basel; 2014 - 68.
Amado E, Schöps R, Brandt W, Kressler J. Spontaneous formation of giant bioactive protein-block copolymer vesicles in water. ACS Macro Letters. 2012; 1 :1016-1019 - 69.
Noor M, Dworeck T, Schenk A, Shinde P, Fioroni M, Schwaneberg U. Polymersome surface decoration by an EGFP fusion protein employing Cecropin a as peptide “anchor”. Journal of Biotechnology. 2012; 157 :31-37 - 70.
Kuang L, Fernandes DA, O’Halloran M, Zheng W, Jiang Y, Ladizhansky V, Brown LS, Liang H. “Frozen” block copolymer nanomembranes with light-driven proton pumping performance. ACS Nano. 2014; 8 :537-545 - 71.
Hua D, Kuang L, Liang H. Self-directed reconstitution of proteorhodopsin with amphiphilic block copolymers induces the formation of hierarchically ordered proteopolymer membrane arrays. Journal of the American Chemical Society. 2011; 133 :2354-2357 - 72.
Kuang L, Olson TL, Lin S, Flores M, Jiang Y, Zheng W, Williams JC, Allen JP, Liang H. Interface for light-driven electron transfer by photosynthetic complexes across block copolymer membranes. Journal of Physical Chemistry Letters. 2014; 5 :787-791 - 73.
Andreasson-Ochsner M, Fu Z, May S, Ying Xiu L, Nallani M, Sinner EK. Selective deposition and self-assembly of triblock copolymers into matrix arrays for membrane protein production. Langmuir. 2012; 28 :2044-2048 - 74.
Gulati S, Jamshad M, Knowles TJ, Morrison KA, Downing R, Cant N, Collins R, Koenderink JB, Ford RC, Overduin M, et al. Detergent-free purification of ABC (ATPbinding-cassette) transporters. The Biochemical Journal. 2014; 461 :269-278 - 75.
Knowles TJ, Finka R, Smith C, Lin YP, Dafforn T, Overduin M. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. Journal of the American Chemical Society. 2009; 131 :7484-7485 - 76.
Hall AR, Scott A, Rotem D, Mehta KK, Bayley H, Dekker C. Hybrid pore formation by directed insertion of α-haemolysin into solid-state nanopores. Nature Nanotechnology. 2010; 5 :874-877 - 77.
Balme S, Janot JM, Berardo L, Henn F, Bonhenry D, Kraszewski S, Picaud F, Ramseyer C. New bioinspired membrane made of a biological ion channel confined into the cylindrical nanopore of a solid-state polymer. Nano Letters. 2011; 11 :712-716 - 78.
Ibragimova S. Supporting and stabilizing biomimetic membranes. Ph.D. Thesis. Lyngby, Denmark: Danish Technical University, Kgs; 2011 - 79.
Mech-Dorosz A, Heiskanen A, Bäckström S, Perry M, Muhammad HB, Hélix-Nielsen C, Emnéus J. A reusable device for electrochemical applications of hydrogel supported black lipid membranes. Biomedical Microdevices. 2015; 17 :21 - 80.
Bodor S, Zook JM, Lindner E, Tóth K, Gyurcsányi RE. Electrochemical methods for the determination of the diffusion coefficient of ionophores and ionophore-ion complexes in plasticized PVC membranes. Analyst. 2008; 133 :635-642 - 81.
Discher DE. Polymer vesicles. Science. 2002; 297 :967-973 - 82.
Nardin C, Hirt T, Leukel J, Meier W. Polymerized ABA triblock copolymer vesicles. Langmuir. 2000; 16 :1035-1041 - 83.
Erbakan M, Shen YX, Grzelakowski M, Butler PJ, Kumar M, Curtis WR. Molecular cloning, overexpression and characterization of a novel water channel protein from rhodobacter sphaeroides. PLoS One. 2014; 9 :e86830 - 84.
Ibata K, Takimoto S, Morisaku T, Miyawaki A, Yasui M. Analysis of aquaporin-mediated diffusional water permeability by coherent anti-stokes raman scattering microscopy. Biophysical Journal. 2011; 101 :2277-2283 - 85.
Mamonov AB, Coalson RD, Zeidel ML, Mathai JC. Water and deuterium oxide permeability through aquaporin 1: MD predictions and experimental verification. The Journal of General Physiology. 2007; 130 :111-116 - 86.
Itel F, Al-Samir S, Oberg F, Chami M, Kumar M, Supuran CT, Deen PMT, Meier W, Hedfalk K, Gros G, Endeward V. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels. The FASEB Journal. 2012; 26 :5182-5191