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Taming membranes: functional immobilization of biological membranes in hydrogels.

ABSTRACT: Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

SUBMITTER: Kusters I

PROVIDER: S-EPMC3105061 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Taming membranes: functional immobilization of biological membranes in hydrogels.

Kusters Ilja I Mukherjee Nobina N de Jong Menno R MR Tans Sander S Koçer Armağan A Driessen Arnold J M AJ

PloS one 20110531 5

Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. ...[more]

PMID: 21655266

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Project description:The high porosity, interconnected pore structure, and high surface area-to-volume ratio make the hydrophilic nonwoven nanofiber membranes (NV-NF-Ms) promising nanostructured supports for enzyme immobilization in different biotechnological applications. In this work, NV-NF-Ms with excellent mechanical and chemical properties were designed and fabricated by electrospinning in one step without using additives or complicated crosslinking processes after electrospinning. To do so, two types of ultrahigh-molecular-weight linear copolymers with very different mechanical properties were used. Methyl methacrylate-co-hydroxyethyl methacrylate (p(MMA)-co-p(HEMA)) and methyl acrylate-co-hydroxyethyl acrylate (p(MA)-co-p(HEA)) were designed and synthesized by reverse atom transfer radical polymerization (reverse-ATRP) and copper-mediated living radical polymerization (Cu0-MC-LRP), respectively. The copolymers were characterized by nuclear magnetic resonance (1H-NMR) spectroscopy and by triple detection gel permeation chromatography (GPC). The polarity, topology, and molecular weight of the copolymers were perfectly adjusted. The polymeric blend formed by (MMA)1002-co-(HEMA)1002 (M w = 230,855 ± 7418 Da; M n = 115,748 ± 35,567 Da; PDI = 2.00) and (MA)11709-co-(HEA)7806 (M w = 1.972 × 106 ± 33,729 Da; M n = 1.395 × 106 ± 35,019 Da; PDI = 1.41) was used to manufacture (without additives or chemical crosslinking processes) hydroxylated nonwoven nanofiber membranes (NV-NF-Ms-OH; 300 nm in fiber diameter) with excellent mechanical and chemical properties. The morphology of NV-NF-Ms-OH was studied by scanning electron microscopy (SEM). The suitability for enzyme binding was proven by designing a palette of different surface functionalization to enable both reversible and irreversible enzyme immobilization. NV-NF-Ms-OH were successfully functionalized with vinyl sulfone (281 ± 20 μmol/g), carboxyl (560 ± 50 μmol/g), and amine groups (281 ± 20 μmol/g) and applied for the immobilization of two enzymes of biotechnological interest. Galactose oxidase was immobilized on vinyl sulfone-activated materials and carboxyl-activated materials, while laccase was immobilized onto amine-activated materials. These preliminary results are a promising basis for the application of nonwoven membranes in enzyme technology.

Dataset Information

Taming membranes: functional immobilization of biological membranes in hydrogels.

Publications

Taming membranes: functional immobilization of biological membranes in hydrogels.

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