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From genes to protein mechanics on a chip.

Otten M, Ott W, Jobst MA, Milles LF, Verdorfer T, Pippig DA, Nash MA, Gaub HE - Nat. Methods (2014)

Bottom Line: Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel.A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever.The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

View Article: PubMed Central - PubMed

Affiliation: 1] Lehrstuhl für Angewandte Physik, Ludwig-Maximilians-Universität, Munich, Germany. [2] Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Munich, Germany. [3].

ABSTRACT
Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

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Unfolding and rupture statistics from multiple force traces.(a-d) relative frequency of observing given contour lengths determined by transforming and aligning multiple force traces into contour length space via the WLC model. Shown are diagrams for the fibronectin tetramer (a) (n = 27, ΔLcFBN = 33 nm), spectrin dimer (b) (n = 50, ΔLcSPN = 34 nm), xylanase monomer (c) (n = 91, ΔLcXYL = 93 nm) and sfGFP monomer (d) (n = 25, ΔLcGFP = 79 nm). (e) Rupture force vs. loading rate scatter plot of final Cohesin-Dockerin dissociation event. (f) Unfolding force vs. loading rate scatter plot for each protein of interest. The populations in e and f were fitted with 2D Gaussians. Respective means and s.d. are plotted in the corresponding colors as solid symbols and error bars.
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Figure 3: Unfolding and rupture statistics from multiple force traces.(a-d) relative frequency of observing given contour lengths determined by transforming and aligning multiple force traces into contour length space via the WLC model. Shown are diagrams for the fibronectin tetramer (a) (n = 27, ΔLcFBN = 33 nm), spectrin dimer (b) (n = 50, ΔLcSPN = 34 nm), xylanase monomer (c) (n = 91, ΔLcXYL = 93 nm) and sfGFP monomer (d) (n = 25, ΔLcGFP = 79 nm). (e) Rupture force vs. loading rate scatter plot of final Cohesin-Dockerin dissociation event. (f) Unfolding force vs. loading rate scatter plot for each protein of interest. The populations in e and f were fitted with 2D Gaussians. Respective means and s.d. are plotted in the corresponding colors as solid symbols and error bars.

Mentions: As a validation and demonstration of our SMFS-MITOMI approach, we expressed genes of interest comprising well-known fingerprint domains in the SMFS literature. We produced multimeric polyproteins including tetrameric human type-III fibronectin (FBN)22 and dimeric chicken brain α-spectrin (SPN)23. We also synthesized monomers of endo-1,4-xylanase T6 from Geobacillus stearothermophilus (XYL)21, superfolder green fluorescent protein (GFP)24, and twitchin kinase25. In all cases, surface immobilization and SMFS assay were enabled by N-terminal ybbR and C-terminal Doc tags on the target proteins. Unfolding data for FBN, SPN, XYL and GFP were obtained with a single cantilever on a single microarray (Figs. 2 and 3). Twitchin kinase was found not to express in sufficient yield to provide reliable unfolding statistics.


From genes to protein mechanics on a chip.

Otten M, Ott W, Jobst MA, Milles LF, Verdorfer T, Pippig DA, Nash MA, Gaub HE - Nat. Methods (2014)

Unfolding and rupture statistics from multiple force traces.(a-d) relative frequency of observing given contour lengths determined by transforming and aligning multiple force traces into contour length space via the WLC model. Shown are diagrams for the fibronectin tetramer (a) (n = 27, ΔLcFBN = 33 nm), spectrin dimer (b) (n = 50, ΔLcSPN = 34 nm), xylanase monomer (c) (n = 91, ΔLcXYL = 93 nm) and sfGFP monomer (d) (n = 25, ΔLcGFP = 79 nm). (e) Rupture force vs. loading rate scatter plot of final Cohesin-Dockerin dissociation event. (f) Unfolding force vs. loading rate scatter plot for each protein of interest. The populations in e and f were fitted with 2D Gaussians. Respective means and s.d. are plotted in the corresponding colors as solid symbols and error bars.
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Related In: Results  -  Collection

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Figure 3: Unfolding and rupture statistics from multiple force traces.(a-d) relative frequency of observing given contour lengths determined by transforming and aligning multiple force traces into contour length space via the WLC model. Shown are diagrams for the fibronectin tetramer (a) (n = 27, ΔLcFBN = 33 nm), spectrin dimer (b) (n = 50, ΔLcSPN = 34 nm), xylanase monomer (c) (n = 91, ΔLcXYL = 93 nm) and sfGFP monomer (d) (n = 25, ΔLcGFP = 79 nm). (e) Rupture force vs. loading rate scatter plot of final Cohesin-Dockerin dissociation event. (f) Unfolding force vs. loading rate scatter plot for each protein of interest. The populations in e and f were fitted with 2D Gaussians. Respective means and s.d. are plotted in the corresponding colors as solid symbols and error bars.
Mentions: As a validation and demonstration of our SMFS-MITOMI approach, we expressed genes of interest comprising well-known fingerprint domains in the SMFS literature. We produced multimeric polyproteins including tetrameric human type-III fibronectin (FBN)22 and dimeric chicken brain α-spectrin (SPN)23. We also synthesized monomers of endo-1,4-xylanase T6 from Geobacillus stearothermophilus (XYL)21, superfolder green fluorescent protein (GFP)24, and twitchin kinase25. In all cases, surface immobilization and SMFS assay were enabled by N-terminal ybbR and C-terminal Doc tags on the target proteins. Unfolding data for FBN, SPN, XYL and GFP were obtained with a single cantilever on a single microarray (Figs. 2 and 3). Twitchin kinase was found not to express in sufficient yield to provide reliable unfolding statistics.

Bottom Line: Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel.A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever.The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

View Article: PubMed Central - PubMed

Affiliation: 1] Lehrstuhl für Angewandte Physik, Ludwig-Maximilians-Universität, Munich, Germany. [2] Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Munich, Germany. [3].

ABSTRACT
Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

Show MeSH
Related in: MedlinePlus