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. 2017 Aug 8;7:7540. doi: 10.1038/s41598-017-08021-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Modulation of the monomer-trimer equilibrium of HA. (A) The cloned HA construct with HA1, HA2 and the foldon, showing mutation sites with arrows and a disulfide bond between HA1 and HA2 (upper panel). The mutation sites are highlighted in red on the crystal structure of CU44 HA at the center with cross-section views along the dashed lines at the left (S199F, V91W and F88E) and right (R75K, R106E and G47E) (middle panel). The amino acid sequence differences at the mutation sites among 2009 pH1N1 and seasonal H1N1 HA proteins are shown (lower panel), where the amino acid residue is numbered according to KR01 HA and the corresponding amino acid residue of HA0 is given in parenthesis. The CU44 wild-type HA in this study showed 3 mutations different from the original CU44 HA: HA1 M116I and HA2 I91V and N169S. (B) Schematic diagrams showing a modulation of the monomer-trimer equilibrium upon the removal of the foldon by thrombin treatment (upper panel). The equilibrium can depend on mutation at the intermonomer interface. The HA trimer where each monomer is shown in blue, red and gray color is dissociated into monomers, if the mutation at the intermonomer interface induces either electrostatic or steric repulsion to destabilize the trimer. Characterization of HA mutant proteins using site exclusion chromatographic analysis (lower panel). F88E and V91W mutants were eluted as a monomer, whereas other mutants were eluted as the wild-type HA trimer. Native PAGE is shown in inset for the pre- and post-thrombin F88E mutant HA. (C) Characterization of the single mutant, F88E and V91W, and the double mutant F88E/V91W using size exclusion chromatography (upper and lower panels, respectively). Single and double mutants without the foldon (post-thrombin) were eluted as monomers. The wild-type HA trimer in blue is shown as a single peak (dashed line).