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Taming membranes: functional immobilization of biological membranes in hydrogels.

Kusters I, Mukherjee N, de Jong MR, Tans S, Koçer A, Driessen AJ - PLoS ONE (2011)

Bottom Line: Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active.Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs.This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, AG Groningen, The Netherlands.

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.

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Hydrogels composed of self-assembling organic gelators immobilize membrane vesicles.(A) Gelator molecule 1 is based on 1,3,5-cyclohexyltricarboxamide and self-assembles into fibers of 20–100 nm. (B) Schematic representation of a hydrogel immobilized GUV with embedded MscL channel. The lipid bilayer is impermeable for the soluble fluorophore calcein when MscL occupies the closed conformation. Addition of MTSET triggers the opening of genetically engineered MscL and allows influx of calcein into the lumen of the GUV. (C) In vitro protein translocation into hydrogel immobilized PLs. SecA translocates the fluorescently labeled preprotein proOmpA through the SecYEG channel by multiple cycles of ATP hydrolysis. Inside the PL, proOmpA is protected against an externally added protease. Molecules in (B) and (C) are not drawn in scale.
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pone-0020435-g001: Hydrogels composed of self-assembling organic gelators immobilize membrane vesicles.(A) Gelator molecule 1 is based on 1,3,5-cyclohexyltricarboxamide and self-assembles into fibers of 20–100 nm. (B) Schematic representation of a hydrogel immobilized GUV with embedded MscL channel. The lipid bilayer is impermeable for the soluble fluorophore calcein when MscL occupies the closed conformation. Addition of MTSET triggers the opening of genetically engineered MscL and allows influx of calcein into the lumen of the GUV. (C) In vitro protein translocation into hydrogel immobilized PLs. SecA translocates the fluorescently labeled preprotein proOmpA through the SecYEG channel by multiple cycles of ATP hydrolysis. Inside the PL, proOmpA is protected against an externally added protease. Molecules in (B) and (C) are not drawn in scale.

Mentions: In this study we present, for the first time, hydrogels composed of organic gelators for the functional immobilization of membrane proteins both in their native lipid environment and in synthetic lipid environments (PLs, GUVs). Hydrogels composed of self-assembling units of low-molecular-weight gelators based on 1,3,5-cyclohexyltricarboxamide form networks of nano-scaled fibers that are an attractive way to immobilize membrane vesicles [14]. The di-ethylene glycol functionalization of the gelator creates a low interacting fiber surface that minimizes surface interactions with biological molecules (Figure 1). Moreover, as fiber-fiber interactions are weak, the local mesh size of the gel is adjusted by the vesicles, allowing them to form their own cavity. Importantly, the vesicles are completely surrounded by an aqueous environment and the self-adapting mesh size of the gel allows the free diffusion of macromolecules such as proteins, yet restricts the movement of the membrane vesicle. The fibers of hydrogelators from the same family of gelators were previously shown to immobilize bilayer liposomes composed of zwitterionic synthetic lipids without interacting at the molecular level [14]. Here, we demonstrate that hydrogels based on 1,3,5-cyclohexyltricarboxamide effectively immobilize inverted membrane vesicles (IMVs) from E. coli, proteoliposomes (PLs) from native E. coli lipids and even several micron sized GUVs composed of a synthetic lipid mixture. The integrity of the lipid bilayer of the different vesicles remains intact during the immobilization procedure. Importantly, the embedded membrane proteins MscL, the mechanosensitive channel of large conductance of E. coli, and SecYEG, the protein-conducting channel of bacteria, are fully active. Since these hydrogels are optically transparent, they allow for fluorescent investigations of the activity of the membrane proteins and processes at the membrane interface down to the single molecule level.


Taming membranes: functional immobilization of biological membranes in hydrogels.

Kusters I, Mukherjee N, de Jong MR, Tans S, Koçer A, Driessen AJ - PLoS ONE (2011)

Hydrogels composed of self-assembling organic gelators immobilize membrane vesicles.(A) Gelator molecule 1 is based on 1,3,5-cyclohexyltricarboxamide and self-assembles into fibers of 20–100 nm. (B) Schematic representation of a hydrogel immobilized GUV with embedded MscL channel. The lipid bilayer is impermeable for the soluble fluorophore calcein when MscL occupies the closed conformation. Addition of MTSET triggers the opening of genetically engineered MscL and allows influx of calcein into the lumen of the GUV. (C) In vitro protein translocation into hydrogel immobilized PLs. SecA translocates the fluorescently labeled preprotein proOmpA through the SecYEG channel by multiple cycles of ATP hydrolysis. Inside the PL, proOmpA is protected against an externally added protease. Molecules in (B) and (C) are not drawn in scale.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105061&req=5

pone-0020435-g001: Hydrogels composed of self-assembling organic gelators immobilize membrane vesicles.(A) Gelator molecule 1 is based on 1,3,5-cyclohexyltricarboxamide and self-assembles into fibers of 20–100 nm. (B) Schematic representation of a hydrogel immobilized GUV with embedded MscL channel. The lipid bilayer is impermeable for the soluble fluorophore calcein when MscL occupies the closed conformation. Addition of MTSET triggers the opening of genetically engineered MscL and allows influx of calcein into the lumen of the GUV. (C) In vitro protein translocation into hydrogel immobilized PLs. SecA translocates the fluorescently labeled preprotein proOmpA through the SecYEG channel by multiple cycles of ATP hydrolysis. Inside the PL, proOmpA is protected against an externally added protease. Molecules in (B) and (C) are not drawn in scale.
Mentions: In this study we present, for the first time, hydrogels composed of organic gelators for the functional immobilization of membrane proteins both in their native lipid environment and in synthetic lipid environments (PLs, GUVs). Hydrogels composed of self-assembling units of low-molecular-weight gelators based on 1,3,5-cyclohexyltricarboxamide form networks of nano-scaled fibers that are an attractive way to immobilize membrane vesicles [14]. The di-ethylene glycol functionalization of the gelator creates a low interacting fiber surface that minimizes surface interactions with biological molecules (Figure 1). Moreover, as fiber-fiber interactions are weak, the local mesh size of the gel is adjusted by the vesicles, allowing them to form their own cavity. Importantly, the vesicles are completely surrounded by an aqueous environment and the self-adapting mesh size of the gel allows the free diffusion of macromolecules such as proteins, yet restricts the movement of the membrane vesicle. The fibers of hydrogelators from the same family of gelators were previously shown to immobilize bilayer liposomes composed of zwitterionic synthetic lipids without interacting at the molecular level [14]. Here, we demonstrate that hydrogels based on 1,3,5-cyclohexyltricarboxamide effectively immobilize inverted membrane vesicles (IMVs) from E. coli, proteoliposomes (PLs) from native E. coli lipids and even several micron sized GUVs composed of a synthetic lipid mixture. The integrity of the lipid bilayer of the different vesicles remains intact during the immobilization procedure. Importantly, the embedded membrane proteins MscL, the mechanosensitive channel of large conductance of E. coli, and SecYEG, the protein-conducting channel of bacteria, are fully active. Since these hydrogels are optically transparent, they allow for fluorescent investigations of the activity of the membrane proteins and processes at the membrane interface down to the single molecule level.

Bottom Line: Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active.Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs.This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, AG Groningen, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus