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Long-term storage of surface-adsorbed protein machines.

Albet-Torres N, Månsson A - Langmuir (2011)

Bottom Line: The effective and simple long-term storage of complex functional proteins is critical in achieving commercially viable biosensors.Importantly, therefore, we here describe that delicate heavy meromyosin (HMM)-based nanodevices (HMM motor fragments adsorbed to silanized surfaces and actin bound to HMM) fully maintain their function when stored at -20 °C for more than a month.The results are important to the future commercial implementation of motor-based nanodevices and are of more general value to the long-term storage of any protein-based bionanodevice.

View Article: PubMed Central - PubMed

Affiliation: School of Natural Sciences, Linnaeus University, SE-39245 Kalmar, Sweden. nuria.albettorres@lnu.se

ABSTRACT
The effective and simple long-term storage of complex functional proteins is critical in achieving commercially viable biosensors. This issue is particularly challenging in recently proposed types of nanobiosensors, where molecular-motor-driven transportation substitutes microfluidics and forms the basis for novel detection schemes. Importantly, therefore, we here describe that delicate heavy meromyosin (HMM)-based nanodevices (HMM motor fragments adsorbed to silanized surfaces and actin bound to HMM) fully maintain their function when stored at -20 °C for more than a month. The mechanisms for the excellent preservation of acto-HMM motor function upon repeated freeze-thaw cycles are discussed. The results are important to the future commercial implementation of motor-based nanodevices and are of more general value to the long-term storage of any protein-based bionanodevice.

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Actin filament path traces are integrated for 10 s: (A) the flow cell before freezing (same sequence as shown in Movie 1 in the Supporting Information), (B) the same flow cell after having been frozen for 17 days and thawed without adding any new solution (the same as in Movie 2), and (C) the same flow cell after adding new actin and ATP-containing solution (the same as in Movie 3). White bars, 10 μm. Yellow parts of the traces indicate the filament position 0, 3.2, and 6.8 s after the onset of recording.
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fig1: Actin filament path traces are integrated for 10 s: (A) the flow cell before freezing (same sequence as shown in Movie 1 in the Supporting Information), (B) the same flow cell after having been frozen for 17 days and thawed without adding any new solution (the same as in Movie 2), and (C) the same flow cell after adding new actin and ATP-containing solution (the same as in Movie 3). White bars, 10 μm. Yellow parts of the traces indicate the filament position 0, 3.2, and 6.8 s after the onset of recording.

Mentions: The HMM-propelled actin filament motility was maintained after the freezing and thawing of whole flow cells that contained all of the components of the IVMA (Experimental Section). When the flow cell had been frozen with both actin filaments and an ATP-containing assay solution and stored at −20 °C for up to 30 days, motility was resumed after thawing without the addition of new assay solution. This is exemplified in Figure 1A–C (Supporting Information, Movie 2). However, for quantitative comparison to the prefreezing conditions, it was important to avoid complications attributed to photobleaching, the consumption of ATP, and the accumulation of possible toxic products (e.g., gluconic acid due to the antibleaching mixture) during the first observation in the fluorescence microscope (before freezing). We therefore added a new assay solution and new actin filaments prior to quantitative measurements (Figure 1C). It can be seen in Figure 2 that both the sliding velocity and the fraction of motile filaments were similar to the values before freezing (no significant difference; p = 0.08 (velocity), p = 0.13 (fraction motile)). Data from different experiments was pooled in a single diagram because there was no apparent difference between flow cells kept frozen for different time periods (up to 30 days). Thus, motility was maintained for more than 1 month of freezing without a noticeable decay over this time period (Figures 3 and 4). In contrast to what was reported for lyophilization and critical-point drying(20) and subsequent reconstitution of the kinesin–microtubule motility assays, we did not observe any appreciable freeze–thaw-induced variability in function between different regions of a flow cell. Because we did not test lyophilization or other drying methods, these approaches might not be ideal for actomyosin (cf. ref (24)).


Long-term storage of surface-adsorbed protein machines.

Albet-Torres N, Månsson A - Langmuir (2011)

Actin filament path traces are integrated for 10 s: (A) the flow cell before freezing (same sequence as shown in Movie 1 in the Supporting Information), (B) the same flow cell after having been frozen for 17 days and thawed without adding any new solution (the same as in Movie 2), and (C) the same flow cell after adding new actin and ATP-containing solution (the same as in Movie 3). White bars, 10 μm. Yellow parts of the traces indicate the filament position 0, 3.2, and 6.8 s after the onset of recording.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104519&req=5

fig1: Actin filament path traces are integrated for 10 s: (A) the flow cell before freezing (same sequence as shown in Movie 1 in the Supporting Information), (B) the same flow cell after having been frozen for 17 days and thawed without adding any new solution (the same as in Movie 2), and (C) the same flow cell after adding new actin and ATP-containing solution (the same as in Movie 3). White bars, 10 μm. Yellow parts of the traces indicate the filament position 0, 3.2, and 6.8 s after the onset of recording.
Mentions: The HMM-propelled actin filament motility was maintained after the freezing and thawing of whole flow cells that contained all of the components of the IVMA (Experimental Section). When the flow cell had been frozen with both actin filaments and an ATP-containing assay solution and stored at −20 °C for up to 30 days, motility was resumed after thawing without the addition of new assay solution. This is exemplified in Figure 1A–C (Supporting Information, Movie 2). However, for quantitative comparison to the prefreezing conditions, it was important to avoid complications attributed to photobleaching, the consumption of ATP, and the accumulation of possible toxic products (e.g., gluconic acid due to the antibleaching mixture) during the first observation in the fluorescence microscope (before freezing). We therefore added a new assay solution and new actin filaments prior to quantitative measurements (Figure 1C). It can be seen in Figure 2 that both the sliding velocity and the fraction of motile filaments were similar to the values before freezing (no significant difference; p = 0.08 (velocity), p = 0.13 (fraction motile)). Data from different experiments was pooled in a single diagram because there was no apparent difference between flow cells kept frozen for different time periods (up to 30 days). Thus, motility was maintained for more than 1 month of freezing without a noticeable decay over this time period (Figures 3 and 4). In contrast to what was reported for lyophilization and critical-point drying(20) and subsequent reconstitution of the kinesin–microtubule motility assays, we did not observe any appreciable freeze–thaw-induced variability in function between different regions of a flow cell. Because we did not test lyophilization or other drying methods, these approaches might not be ideal for actomyosin (cf. ref (24)).

Bottom Line: The effective and simple long-term storage of complex functional proteins is critical in achieving commercially viable biosensors.Importantly, therefore, we here describe that delicate heavy meromyosin (HMM)-based nanodevices (HMM motor fragments adsorbed to silanized surfaces and actin bound to HMM) fully maintain their function when stored at -20 °C for more than a month.The results are important to the future commercial implementation of motor-based nanodevices and are of more general value to the long-term storage of any protein-based bionanodevice.

View Article: PubMed Central - PubMed

Affiliation: School of Natural Sciences, Linnaeus University, SE-39245 Kalmar, Sweden. nuria.albettorres@lnu.se

ABSTRACT
The effective and simple long-term storage of complex functional proteins is critical in achieving commercially viable biosensors. This issue is particularly challenging in recently proposed types of nanobiosensors, where molecular-motor-driven transportation substitutes microfluidics and forms the basis for novel detection schemes. Importantly, therefore, we here describe that delicate heavy meromyosin (HMM)-based nanodevices (HMM motor fragments adsorbed to silanized surfaces and actin bound to HMM) fully maintain their function when stored at -20 °C for more than a month. The mechanisms for the excellent preservation of acto-HMM motor function upon repeated freeze-thaw cycles are discussed. The results are important to the future commercial implementation of motor-based nanodevices and are of more general value to the long-term storage of any protein-based bionanodevice.

Show MeSH