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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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Protein interactions in the hydrogel.A fraction of SecA-FM (A) and proOmpA-Atto647N in complex with the chaperone SecB (B) bind non-specifically to the OG8 fibers. SecA-FM (C) binding to PLs containing SecYEG-Atto647N (D) is visible as co-localizing signal. In presence of SecYEG-PLs non-specific binding of SecA-FM to the gel fibers is eliminated (A,C).
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pone-0020435-g004: Protein interactions in the hydrogel.A fraction of SecA-FM (A) and proOmpA-Atto647N in complex with the chaperone SecB (B) bind non-specifically to the OG8 fibers. SecA-FM (C) binding to PLs containing SecYEG-Atto647N (D) is visible as co-localizing signal. In presence of SecYEG-PLs non-specific binding of SecA-FM to the gel fibers is eliminated (A,C).

Mentions: Next, the compatibility of the hydrogel with fluorescently labeled proteins was tested. Here, we focus on the protein translocation system of E. coli. This system consists of a multi-protein complex termed ’translocase’ that includes a protein conducting channel, SecYEG, embedded in the cytoplasmic membrane and a motor protein, SecA (for review see [22]). Secretory proteins (preproteins) are translocated through the translocase by SecA through multiple cycles of ATP hydrolysis (Figure 1C) [23], [24], [25]. Protein translocation of fluorescently labeled preproteins into SecYEG containing IMVs or PLs can be followed in bulk by protease protection [26] and by the formation of a translocation intermediate that can be monitored by FRET between a donor-fluorophore on the trapped preprotein and an acceptor fluorophore attached to the exit of the SecYEG pore (A. Kedrov et al., submitted). Here, using the procedure described above, we mixed proteins and PLs into hydrogels containing 0.65% gelator 1. While GFP was found freely diffusing in the gel as determined by fluorescence cross correlation spectroscopy (FCS, data not shown), the very hydrophobic and partially unfolded model preprotein proOmpA labeled with Atto647N bound non-specifically to the gel fibers (Figure 4B). Furthermore, a fraction of the otherwise freely diffusing fluorescein labeled SecA (SecA-FM) interacted with the fibers in absence of SecYEG containing PLs (Figure 4A). However, the non-specific binding of SecA-FM was virtually absent in the presence of immobilized proteoliposomes containing SecYEG-Atto647N that bound SecA-FM specifically (compare Figure 4C and D). This is likely due to the lower concentration of soluble SecA as a large fraction of SecA is bound to the SecYEG-PLs suggesting that the non-specific interaction is dependent on the SecA concentration. Altogether, this data demonstrates that interaction of a membrane receptor (SecYEG) with its ligand (SecA) can be studied on single liposome level using hydrogel immobilized PLs or IMVs.


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)

Protein interactions in the hydrogel.A fraction of SecA-FM (A) and proOmpA-Atto647N in complex with the chaperone SecB (B) bind non-specifically to the OG8 fibers. SecA-FM (C) binding to PLs containing SecYEG-Atto647N (D) is visible as co-localizing signal. In presence of SecYEG-PLs non-specific binding of SecA-FM to the gel fibers is eliminated (A,C).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020435-g004: Protein interactions in the hydrogel.A fraction of SecA-FM (A) and proOmpA-Atto647N in complex with the chaperone SecB (B) bind non-specifically to the OG8 fibers. SecA-FM (C) binding to PLs containing SecYEG-Atto647N (D) is visible as co-localizing signal. In presence of SecYEG-PLs non-specific binding of SecA-FM to the gel fibers is eliminated (A,C).
Mentions: Next, the compatibility of the hydrogel with fluorescently labeled proteins was tested. Here, we focus on the protein translocation system of E. coli. This system consists of a multi-protein complex termed ’translocase’ that includes a protein conducting channel, SecYEG, embedded in the cytoplasmic membrane and a motor protein, SecA (for review see [22]). Secretory proteins (preproteins) are translocated through the translocase by SecA through multiple cycles of ATP hydrolysis (Figure 1C) [23], [24], [25]. Protein translocation of fluorescently labeled preproteins into SecYEG containing IMVs or PLs can be followed in bulk by protease protection [26] and by the formation of a translocation intermediate that can be monitored by FRET between a donor-fluorophore on the trapped preprotein and an acceptor fluorophore attached to the exit of the SecYEG pore (A. Kedrov et al., submitted). Here, using the procedure described above, we mixed proteins and PLs into hydrogels containing 0.65% gelator 1. While GFP was found freely diffusing in the gel as determined by fluorescence cross correlation spectroscopy (FCS, data not shown), the very hydrophobic and partially unfolded model preprotein proOmpA labeled with Atto647N bound non-specifically to the gel fibers (Figure 4B). Furthermore, a fraction of the otherwise freely diffusing fluorescein labeled SecA (SecA-FM) interacted with the fibers in absence of SecYEG containing PLs (Figure 4A). However, the non-specific binding of SecA-FM was virtually absent in the presence of immobilized proteoliposomes containing SecYEG-Atto647N that bound SecA-FM specifically (compare Figure 4C and D). This is likely due to the lower concentration of soluble SecA as a large fraction of SecA is bound to the SecYEG-PLs suggesting that the non-specific interaction is dependent on the SecA concentration. Altogether, this data demonstrates that interaction of a membrane receptor (SecYEG) with its ligand (SecA) can be studied on single liposome level using hydrogel immobilized PLs or IMVs.

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