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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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Activity of hydrogel immobilized PL monitored by DCFBA-FRET.Immobilized proteoliposomes containing SecYEG-FM were excited with a 488 nm laser only and fluorescence at 515–545 nm (A, D) and 650–700 nm (B, E) was recorded simultaneously. (A) PLs containing SecYEG-FM after addition of buffer. (B) The same area as (A) at 650–700 nm shows only background fluorescence. (C) Schematic representation of proOmpA-DhfR intermediate formation. In presence of ATP, SecA translocates the proOmpA domain until the tightly folded DhfR domain stalls translocation resulting in a stable complex. Atto647N on proOmpA serves as acceptor for FRET from a donor fluorophore attached to the exit of the SeYEG channel. (D) PLs containing SecYEG-FM after addition of ATP leading to formation of protein translocation intermediates of proOmpA-DhfR-Atto647N. (E) Resulting FRET signals appear as co-localizing fluorescence at 650–700 nm. (F) Quantification of FRET efficiencies by DCFBA in the hydrogel and in suspension.
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pone-0020435-g006: Activity of hydrogel immobilized PL monitored by DCFBA-FRET.Immobilized proteoliposomes containing SecYEG-FM were excited with a 488 nm laser only and fluorescence at 515–545 nm (A, D) and 650–700 nm (B, E) was recorded simultaneously. (A) PLs containing SecYEG-FM after addition of buffer. (B) The same area as (A) at 650–700 nm shows only background fluorescence. (C) Schematic representation of proOmpA-DhfR intermediate formation. In presence of ATP, SecA translocates the proOmpA domain until the tightly folded DhfR domain stalls translocation resulting in a stable complex. Atto647N on proOmpA serves as acceptor for FRET from a donor fluorophore attached to the exit of the SeYEG channel. (D) PLs containing SecYEG-FM after addition of ATP leading to formation of protein translocation intermediates of proOmpA-DhfR-Atto647N. (E) Resulting FRET signals appear as co-localizing fluorescence at 650–700 nm. (F) Quantification of FRET efficiencies by DCFBA in the hydrogel and in suspension.

Mentions: Since the chaperone SecB keeps the model substrate proOmpA in a partially unfolded and therefore translocation competent state, substantial non specific interactions of proOmpA with membranes and other surfaces occur. This renders monitoring of protein translocation by means of proOmpA accumulation inside of small vesicles intrinsically difficult. Therefore, to quantitatively assess protein translocation activity at the single liposome level, we performed DCFBA-FRET experiments employing a dual color laser scanning confocal microscope. As proOmpA was labeled with the acceptor fluorophore Atto647N (647 nm/669 nm, excitation/emission) non specifically bound proOmpA was not visible when exciting the donor fluorophore with the 488 nm laser line. The ratio of donor and acceptor fluorescence intensity in a FRET experiment can be used to determine FRET efficiencies and by immobilizing PLs in the hydrogel, FRET efficiencies for individual liposomes can be obtained. Proteoliposomes containing fluorescein labeled SecYEG, SecA and the fusion protein proOmpA(C282)-DhfR-Atto647N in complex with SecB were immobilized in a hydrogel of gelator 1 as described in the previous sections (Figure 6). The DhfR domain of the proOmpA-DhfR fusion protein is tightly folded in presence of its ligands NADPH and methotraxate and translocation results in a stable translocation intermediate with the proOmpA-DhfR trapped in the SecYEG channel (Figure 6C) [27], [28]. Using fluorescently labeled preprotein and SecY, formation of the translocation intermediate is expected to result in FRET between the donor fluorophore on SecYEG and the acceptor on proOmpA(C282)-DhfR (Kedrov et al, submitted, Figure 6C). In order to monitor single liposome FRET of translocation intermediates, we performed confocal scans in the gel to record fluorescence signals for blue (donor fluorophore on SecY, Figure 6A and D) and red fluorescence (acceptor fluorophore on proOmpA-DhfR, Figure 6B and E) simultaneously, using the 488 nm laser only for donor excitation. Co-localizing signals were identified and quantified with DCFBA as the ratio of proOmpA (C282)-DhfR-Atto647N over SecYEG-FM fluorescence. This ratio serves as an arbitrary unit for the FRET efficiency per liposome. In absence of ATP, no ratio of Atto647N/FM could be determined as fluorescence intensities at 650–700 nm did not exceed background levels (Figure 6B and F). However, when ATP was added to the reaction, fluorescent signals at 650–700 nm appeared originating from FRET between fluorophores on SecY and proOmpA-DhfR (compare Figure 6D and E, respectively). The efficiency of translocation intermediate formation in the hydrogel as determined by the average FRET efficiency per liposome was ∼50% compared to suspension (Figure 6F). This further demonstrates that the translocon is functional when reconstituted in PLs that are immobilized in the hydrogel.


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)

Activity of hydrogel immobilized PL monitored by DCFBA-FRET.Immobilized proteoliposomes containing SecYEG-FM were excited with a 488 nm laser only and fluorescence at 515–545 nm (A, D) and 650–700 nm (B, E) was recorded simultaneously. (A) PLs containing SecYEG-FM after addition of buffer. (B) The same area as (A) at 650–700 nm shows only background fluorescence. (C) Schematic representation of proOmpA-DhfR intermediate formation. In presence of ATP, SecA translocates the proOmpA domain until the tightly folded DhfR domain stalls translocation resulting in a stable complex. Atto647N on proOmpA serves as acceptor for FRET from a donor fluorophore attached to the exit of the SeYEG channel. (D) PLs containing SecYEG-FM after addition of ATP leading to formation of protein translocation intermediates of proOmpA-DhfR-Atto647N. (E) Resulting FRET signals appear as co-localizing fluorescence at 650–700 nm. (F) Quantification of FRET efficiencies by DCFBA in the hydrogel and in suspension.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105061&req=5

pone-0020435-g006: Activity of hydrogel immobilized PL monitored by DCFBA-FRET.Immobilized proteoliposomes containing SecYEG-FM were excited with a 488 nm laser only and fluorescence at 515–545 nm (A, D) and 650–700 nm (B, E) was recorded simultaneously. (A) PLs containing SecYEG-FM after addition of buffer. (B) The same area as (A) at 650–700 nm shows only background fluorescence. (C) Schematic representation of proOmpA-DhfR intermediate formation. In presence of ATP, SecA translocates the proOmpA domain until the tightly folded DhfR domain stalls translocation resulting in a stable complex. Atto647N on proOmpA serves as acceptor for FRET from a donor fluorophore attached to the exit of the SeYEG channel. (D) PLs containing SecYEG-FM after addition of ATP leading to formation of protein translocation intermediates of proOmpA-DhfR-Atto647N. (E) Resulting FRET signals appear as co-localizing fluorescence at 650–700 nm. (F) Quantification of FRET efficiencies by DCFBA in the hydrogel and in suspension.
Mentions: Since the chaperone SecB keeps the model substrate proOmpA in a partially unfolded and therefore translocation competent state, substantial non specific interactions of proOmpA with membranes and other surfaces occur. This renders monitoring of protein translocation by means of proOmpA accumulation inside of small vesicles intrinsically difficult. Therefore, to quantitatively assess protein translocation activity at the single liposome level, we performed DCFBA-FRET experiments employing a dual color laser scanning confocal microscope. As proOmpA was labeled with the acceptor fluorophore Atto647N (647 nm/669 nm, excitation/emission) non specifically bound proOmpA was not visible when exciting the donor fluorophore with the 488 nm laser line. The ratio of donor and acceptor fluorescence intensity in a FRET experiment can be used to determine FRET efficiencies and by immobilizing PLs in the hydrogel, FRET efficiencies for individual liposomes can be obtained. Proteoliposomes containing fluorescein labeled SecYEG, SecA and the fusion protein proOmpA(C282)-DhfR-Atto647N in complex with SecB were immobilized in a hydrogel of gelator 1 as described in the previous sections (Figure 6). The DhfR domain of the proOmpA-DhfR fusion protein is tightly folded in presence of its ligands NADPH and methotraxate and translocation results in a stable translocation intermediate with the proOmpA-DhfR trapped in the SecYEG channel (Figure 6C) [27], [28]. Using fluorescently labeled preprotein and SecY, formation of the translocation intermediate is expected to result in FRET between the donor fluorophore on SecYEG and the acceptor on proOmpA(C282)-DhfR (Kedrov et al, submitted, Figure 6C). In order to monitor single liposome FRET of translocation intermediates, we performed confocal scans in the gel to record fluorescence signals for blue (donor fluorophore on SecY, Figure 6A and D) and red fluorescence (acceptor fluorophore on proOmpA-DhfR, Figure 6B and E) simultaneously, using the 488 nm laser only for donor excitation. Co-localizing signals were identified and quantified with DCFBA as the ratio of proOmpA (C282)-DhfR-Atto647N over SecYEG-FM fluorescence. This ratio serves as an arbitrary unit for the FRET efficiency per liposome. In absence of ATP, no ratio of Atto647N/FM could be determined as fluorescence intensities at 650–700 nm did not exceed background levels (Figure 6B and F). However, when ATP was added to the reaction, fluorescent signals at 650–700 nm appeared originating from FRET between fluorophores on SecY and proOmpA-DhfR (compare Figure 6D and E, respectively). The efficiency of translocation intermediate formation in the hydrogel as determined by the average FRET efficiency per liposome was ∼50% compared to suspension (Figure 6F). This further demonstrates that the translocon is functional when reconstituted in PLs that are immobilized in the hydrogel.

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