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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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In vitro protein translocation in a hydrogel composed of gelator 1 and in suspension.Upon addition of ATP, SecA mediates the translocation of proOmpA into the PLs thereby rendering it resistant against an externally added protease (A). Translocation in the hydrogel or in suspension can be triggered by UV light when NPE-caged ATP is present (B). Lane 5, 6 serves as standard 5 and 10% of input material, respectively.
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pone-0020435-g005: In vitro protein translocation in a hydrogel composed of gelator 1 and in suspension.Upon addition of ATP, SecA mediates the translocation of proOmpA into the PLs thereby rendering it resistant against an externally added protease (A). Translocation in the hydrogel or in suspension can be triggered by UV light when NPE-caged ATP is present (B). Lane 5, 6 serves as standard 5 and 10% of input material, respectively.

Mentions: (A) Addition of MTSET to the top of the hydrogel causes opening of the membrane embedded MscL channel whereupon the soluble fluorophore calcein diffuses into the lumen of the GUV. (B) Calcein fluorescence inside and outside of the GUV quantified by cross-sections through the center of the GUV depicted in (A) at the different time points; t = 0 s (straight line), 45 s (dash-dot), 90 s (dot), 180 s (dash). (C) Calcein fluorescence at the center of the GUV (A) at the indicated time points (straight line). Background fluorescence inside a GUV (as shown in Fig. 5) did not change upon addition of buffer (dashed line).


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)

In vitro protein translocation in a hydrogel composed of gelator 1 and in suspension.Upon addition of ATP, SecA mediates the translocation of proOmpA into the PLs thereby rendering it resistant against an externally added protease (A). Translocation in the hydrogel or in suspension can be triggered by UV light when NPE-caged ATP is present (B). Lane 5, 6 serves as standard 5 and 10% of input material, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020435-g005: In vitro protein translocation in a hydrogel composed of gelator 1 and in suspension.Upon addition of ATP, SecA mediates the translocation of proOmpA into the PLs thereby rendering it resistant against an externally added protease (A). Translocation in the hydrogel or in suspension can be triggered by UV light when NPE-caged ATP is present (B). Lane 5, 6 serves as standard 5 and 10% of input material, respectively.
Mentions: (A) Addition of MTSET to the top of the hydrogel causes opening of the membrane embedded MscL channel whereupon the soluble fluorophore calcein diffuses into the lumen of the GUV. (B) Calcein fluorescence inside and outside of the GUV quantified by cross-sections through the center of the GUV depicted in (A) at the different time points; t = 0 s (straight line), 45 s (dash-dot), 90 s (dot), 180 s (dash). (C) Calcein fluorescence at the center of the GUV (A) at the indicated time points (straight line). Background fluorescence inside a GUV (as shown in Fig. 5) did not change upon addition of buffer (dashed line).

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