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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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Related in: MedlinePlus

Single-molecule force traces recorded in different protein spots on a single chip with a single cantilever.(a-d) Four proteins of interest, anchored between the CoA-functionalized surface and the Cohesin-functionalized cantilever were probed: fibronectin tetramer (a, olive), spectrin dimer (b, red), xylanase monomer (c, blue), and sfGFP monomer (d, green). The crystal structure and pulling configuration (top) are shown for each construct. Each single-molecule force-distance trace (bottom) shows the individual unfolding fingerprint of the respective protein of interest followed by a common, final double sawtooth peak (grey), characteristic of the Cohesin-Dockerin rupture. Experimental data were fitted with the wormlike chain model (dashed lines). Unfolding intermediates were also observed (only fitted for xylanase in c; dotted colored line).
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Figure 2: Single-molecule force traces recorded in different protein spots on a single chip with a single cantilever.(a-d) Four proteins of interest, anchored between the CoA-functionalized surface and the Cohesin-functionalized cantilever were probed: fibronectin tetramer (a, olive), spectrin dimer (b, red), xylanase monomer (c, blue), and sfGFP monomer (d, green). The crystal structure and pulling configuration (top) are shown for each construct. Each single-molecule force-distance trace (bottom) shows the individual unfolding fingerprint of the respective protein of interest followed by a common, final double sawtooth peak (grey), characteristic of the Cohesin-Dockerin rupture. Experimental data were fitted with the wormlike chain model (dashed lines). Unfolding intermediates were also observed (only fitted for xylanase in c; dotted colored line).

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)

Single-molecule force traces recorded in different protein spots on a single chip with a single cantilever.(a-d) Four proteins of interest, anchored between the CoA-functionalized surface and the Cohesin-functionalized cantilever were probed: fibronectin tetramer (a, olive), spectrin dimer (b, red), xylanase monomer (c, blue), and sfGFP monomer (d, green). The crystal structure and pulling configuration (top) are shown for each construct. Each single-molecule force-distance trace (bottom) shows the individual unfolding fingerprint of the respective protein of interest followed by a common, final double sawtooth peak (grey), characteristic of the Cohesin-Dockerin rupture. Experimental data were fitted with the wormlike chain model (dashed lines). Unfolding intermediates were also observed (only fitted for xylanase in c; dotted colored line).
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Related In: Results  -  Collection

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Figure 2: Single-molecule force traces recorded in different protein spots on a single chip with a single cantilever.(a-d) Four proteins of interest, anchored between the CoA-functionalized surface and the Cohesin-functionalized cantilever were probed: fibronectin tetramer (a, olive), spectrin dimer (b, red), xylanase monomer (c, blue), and sfGFP monomer (d, green). The crystal structure and pulling configuration (top) are shown for each construct. Each single-molecule force-distance trace (bottom) shows the individual unfolding fingerprint of the respective protein of interest followed by a common, final double sawtooth peak (grey), characteristic of the Cohesin-Dockerin rupture. Experimental data were fitted with the wormlike chain model (dashed lines). Unfolding intermediates were also observed (only fitted for xylanase in c; dotted colored line).
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