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. 2018 Jun 27;8:9725. doi: 10.1038/s41598-018-27860-w

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© The Author(s) 2018

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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Polyacrylamide gel electrophoresis and ultracentrifugation analysis of wild-type and mutant enzymes. (a) SDS-PAGE (17.5% gel) of purified NylC. Lane M, molecular size marker; Lane 1, NylC_A; Lane 2, NylC_K; Lane 3, NylC_p2; Lane 4, NylC_p2-K¹²²; Lane 5, NylC_p2-L¹²²; Lane 6, NylC_p2-R¹²²; Lane 7, NylC_p2-V¹²²; Lane 8, NylC_p2-Q¹²²; Lane 9, NylC_p2-N¹²²; Lane 10, NylC_p2-G¹²²Y¹³⁰A³⁶Q²⁶³; Lane 11, NylC_p2-A¹³⁷; Lane 12, NylC_p2-G¹²²Y¹³⁰; Lane 13, NylC_p2-G¹²²A¹³⁷; Lane 14, NylC_p2-G¹²²; and Lane 15, NylC_p2-Y¹³⁰. (b) Native-PAGE (10% gel) of purified NylC. The wild-type and most NylC mutants exhibited single bands corresponding to the dimeric structure (shown as “D”). However, some mutants (NylC_p2-A¹³⁷ and NylC_p2-G¹²²A¹³⁷) exhibited a faster-migrating band (presumed to be the monomer and shown as “M”). In contrast, the most thermostable mutant, G¹²²Y¹³⁰A³⁶Q²⁶³, showed the slowest-migrating band (presumed to be tetrameric and shown as “T”). Lane 1, NylC_A; Lane 2, NylC_K; Lane 3, NylC_p2; Lane 4, NylC_p2-L¹²²; Lane 5, NylC_p2-N¹²²; Lane 6, NylC_p2-Q¹²²; Lane 7, NylC_p2-R¹²²; Lane 8, NylC_p2-K¹²²; Lane 9, NylC_p2-V¹²²; Lane 10, NylC_p2-G¹²²; Lane 11, NylC_p2-G¹²²Y¹³⁰A³⁶Q²⁶³; Lane 12, NylC_p2-G¹²²Y¹³⁰; Lane 13, NylC_p2-A¹³⁷; and Lane 14, NylC_p2-G¹²²A¹³⁷. The grouping of portions cropped from different gels was made explicit using delineation with dividing white space. Full-length gels are included in a Supplementary Information file (Fig. S8). (c,d) Ultracentrifugation analysis. The subunit assembly of the wild-type enzymes (NylC_p2, NylC_A, and NylC_K) and mutant enzymes derived from NylC_p2 (G¹²²Y¹³⁰A³⁶Q²⁶³, A¹³⁷, and G¹²²A¹³⁷) was analyzed by sedimentation velocity (c) and sedimentation equilibrium methods (d). The estimated oligomeric states, which are the monomer (M), dimer (D), trimer (Tr), and tetramer (T), for each peak in (c) are marked. The deviations of the s values of the same oligomeric state suggest that the shape of the protein structure (spatial location of loop regions and bulkiness of protein) affects the sedimentation coefficient in aqueous solution. The wild-type NylC_A gave a single peak (4.7 S) corresponding to a tetramer. The wild-type NylC_K gave one major peak (4.3 S) and two minor peaks (7.5 S and 2.1 S), suggesting that the monomer, tetramer, and higher oligomers coexisted in equilibrium. The wild-type NylC_p2 gave two major peaks [3.7 S (trimer) and 3.2 S (dimer)] and a minor peak (2.1 S) (monomer). The G¹²²Y¹³⁰A³⁶Q²⁶³ mutant gave one major peak corresponding to 4.5 S (tetramer) and a minor peak (2.4 S) (monomer). The A¹³⁷ mutant gave a major peak (2.7 S) (dimer) and two minor peaks [2.0 S (monomer) and 5.0 S] molecular species. The G¹²²A¹³⁷ mutant gave a major peak of 2.6 S (monomer or dimer) and a minor peak of 3.5 S (trimer).