Hoffmann, T, Tych, KM, Crosskey, T et al. (3 more authors) (2015) Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of β-Barrel Assembly Machinery Polypeptide Transport Associated Domains. ACS Nano, 9 (9). pp. 8811-8821. ISSN 1936-0851
Abstract
Single-molecule force spectroscopy by atomic force microscopy exploits the use of multimeric protein constructs, namely, polyproteins, to decrease the impact of nonspecific interactions, to improve data accumulation, and to allow the accommodation of benchmarking reference domains within the construct. However, methods to generate such constructs are either time- and labor-intensive or lack control over the length or the domain sequence of the obtained construct. Here, we describe an approach that addresses both of these shortcomings that uses Gibson assembly (GA) to generate a defined recombinant polyprotein rapidly using linker sequences. To demonstrate the feasibility of this approach, we used GA to make a polyprotein composed of alternating domains of I27 and TmCsp, (I27-TmCsp)3-I27)(GA), and showed the mechanical fingerprint, mechanical strength, and pulling speed dependence are the same as an analogous polyprotein constructed using the classical approach. After this benchmarking, we exploited this approach to facilitiate the mechanical characterization of POTRA domain 2 of BamA from E. coli (EcPOTRA2) by assembling the polyprotein (I27-EcPOTRA2)3-I27(GA). We show that, as predicted from the α + β topology, EcPOTRA2 domains are mechanically robust over a wide range of pulling speeds. Furthermore, we identify a clear correlation between mechanical robustness and brittleness for a range of other α + β proteins that contain the structural feature of proximal terminal β-strands in parallel geometry. We thus demonstrate that the GA approach is a powerful tool, as it circumvents the usual time- and labor-intensive polyprotein production process and allows for rapid production of new constructs for single-molecule studies. As shown for EcPOTRA2, this approach allows the exploration of the mechanical properties of a greater number of proteins and their variants. This improves our understanding of the relationship between structure and mechanical strength, increasing our ability to design proteins with tailored mechanical properties.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2015, American Chemical Society. This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Nano, American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/acsnano.5b01962 |
Keywords: | Gibson assembly; polyprotein; force spectroscopy; BamA (β barrel assembly machinery); POTRA (polypeptide transport associated); protein unfolding |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology 2 (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Physics and Astronomy (Leeds) > Molecular & Nanoscale Physics |
Depositing User: | Symplectic Publications |
Date Deposited: | 28 Oct 2015 09:37 |
Last Modified: | 08 Mar 2019 15:38 |
Published Version: | http://dx.doi.org/10.1021/acsnano.5b01962 |
Status: | Published |
Publisher: | American Chemical Society |
Identification Number: | 10.1021/acsnano.5b01962 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:91129 |