Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of β-Barrel Assembly Machinery Polypeptide Transport Associated Domains.
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.
School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, U.K.
Toni
T
Hoffmann
School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, U.K.
Katarzyna M
KM
Tych
School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, U.K.
Thomas
T
Crosskey
School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, U.K.
Bob
B
Schiffrin
School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, U.K.
David J
DJ
Brockwell
School of Physics and Astronomy, ‡Astbury Centre for Structural and Molecular Biology, and §School of Molecular and Cellular Biology, University of Leeds , Leeds, LS2 9JT, U.K.
Lorna
L
Dougan
D004926:Escherichia coli / Q000737:chemistry; D018625:Microscopy, Atomic Force; D010455:Peptides / Q000737:chemistry*; D020815:Polyproteins / Q000737:chemistry* / Q000648:ultrastructure; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
D010455:Peptides; D020815:Polyproteins
BamA (β barrel assembly machinery); Gibson assembly; POTRA (polypeptide transport associated); force spectroscopy; polyprotein; protein unfolding