扭結蛋白是一種特殊的蛋白家族，其可以折疊成不同複雜程度的拓樸結結構。YibK是一種屬於甲基轉移酶家族的三葉型扭結蛋白。為了要研究蛋白扭結對於結構、折疊及功能的重要性，我們基於YibK建構出環形序列重組突變體CP82。YibK的序列被重新排列，其原始的 N- 和 C- 端被連接，並在結上形成新的開口作為新的N- 和C- 端。利用X光晶體繞射及小角度X光散射 (SAXS) 可以證實環形序列重組不會顯著破壞YibK的三維空間結構。儘管總體結構具相似性，藉由圓二色光譜儀及螢光光譜儀偵測到CP82顯現出熱穩定性及化學穩定性的下降。此外，藉由氫氘交換質譜儀可以在高的結構解析度下觀察到CP82的不穩定效應。重要的是，YibK的紐結區域相關於甲基轉移酶活性之輔因子的鍵結，CP82中扭結的解開完全破壞與輔因子鍵結的能力。我們的實驗結果表明，環形序列重組不會破壞YibK的總體結構，但在摺疊穩定性和功能上具有顯著的影響。

Knotted proteins are a special family of proteins that can fold into topological knots with different complexities. YibK is a trefoil-knotted protein belonging to a superfamily of methyltransferases. To investigate the importance of a protein knot to the structure, folding and function, we generated a circular permutant, CP82, based on YibK. The sequence of YibK was rearranged such that the original N- and C- termini were linked and a new opening was introduced at knotting loop to form the new N- and C- termini. The circular permutation (CP) did not significantly perturb the three-dimensional structure of YibK, which was confirmed by X-ray crystallography and small angle X-ray scattering (SAXS). Despite the overall structural similarity, CP82 exhibited a clear reduction in the thermal and chemical stabilities monitored by far-UV CD and intrinsic fluorescence spectroscopy. Furthermore, the destabilization effect of CP82 was observed at a high structural resolution by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Importantly, the knotted region of YibK is responsible for cofactor binding required for its methyltransferase activity. The knotting loop opening in CP82 completely abolished the cofactor binding capacity. Our experimental results indicated that, while CP did not perturb the overall structure of YibK, it generated profound impacts on the folding stability and function.

1. Dabrowski-Tumanski, P. and J. Sulkowska, To Tie or Not to Tie? That Is the Question. Polymers, 2017. 9(12).
2. Richardson, J.S., β-Sheet topology and the relatedness of proteins. Nature, 1977. 268: p. 495.
3. Mansfield, M.L., Are there knots in proteins? Nature Structural Biology, 1994. 1: p. 213.
4. Jamroz, M., et al., KnotProt: a database of proteins with knots and slipknots. Nucleic Acids Res, 2015. 43(Database issue): p. D306-14.
5. Sulkowska, J.I., et al., Conservation of complex knotting and slipknotting patterns in proteins. Proc Natl Acad Sci U S A, 2012. 109(26): p. E1715-23.
6. Thiruselvam, V., et al., Crystal structure analysis of a hypothetical protein (MJ0366) from Methanocaldococcus jannaschii revealed a novel topological arrangement of the knot fold. Biochem Biophys Res Commun, 2017. 482(2): p. 264-269.
7. Nureki, O., et al., An enzyme with a deep trefoil knot for the active-site architecture. Acta Crystallographica Section D, 2002. 58(7): p. 1129-1137.
8. Taylor, W.R., A deeply knotted protein structure and how it might fold. Nature, 2000. 406: p. 916.
9. Bishop, P., D. Rocca, and J.M. Henley, Ubiquitin C-terminal hydrolase L1 (UCH-L1): structure, distribution and roles in brain function and dysfunction. Biochem J, 2016. 473(16): p. 2453-62.
10. Bölinger, D., et al., A Stevedore's Protein Knot. PLOS Computational Biology, 2010. 6(4): p. e1000731.
11. Hori, H., Transfer RNA methyltransferases with a SpoU-TrmD (SPOUT) fold and their modified nucleosides in tRNA. Biomolecules, 2017. 7(1).
12. Anantharaman, V., E.V. Koonin, and L. Aravind, SPOUT: a class of methyltransferases that includes spoU and trmD RNA methylase superfamilies, and novel superfamilies of predicted prokaryotic RNA methylases. J Mol Microbiol Biotechnol, 2002. 4(1): p. 71-5.
13. Mallam, A.L. and S.E. Jackson, The Dimerization of an α/β-Knotted Protein Is Essential for Structure and Function. Structure, 2007. 15(1): p. 111-122.
14. Ito, T., et al., Structural basis for methyl-donor–dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD. Proceedings of the National Academy of Sciences, 2015. 112(31): p. E4197-E4205.
15. Lim, K., et al., Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot. Proteins, 2003. 51(1): p. 56-67.
16. Mallam, A.L. and S.E. Jackson, Folding Studies on a Knotted Protein. Journal of Molecular Biology, 2005. 346(5): p. 1409-1421.
17. Mallam, A. and S. Jackson, Probing Nature's Knots: The Folding Pathway of a Knotted Homodimeric Protein. Vol. 359. 2006. 1420-36.
18. Mallam, A.L. and S.E. Jackson, A comparison of the folding of two knotted proteins: YbeA and YibK. J Mol Biol, 2007. 366(2): p. 650-65.
19. Mallam, A.L., J.M. Rogers, and S.E. Jackson, Experimental detection of knotted conformations in denatured proteins. Proceedings of the National Academy of Sciences, 2010. 107(18): p. 8189-8194.
20. Tkaczuk, K.L., et al., Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases. BMC Bioinformatics, 2007. 8: p. 73.
21. Celniker, G., et al., ConSurf: Using Evolutionary Data to Raise Testable Hypotheses about Protein Function. Israel Journal of Chemistry, 2013. 53(3-4): p. 199-206.
22. Glaser, F., et al., ConSurf: Identification of Functional Regions in Proteins by Surface-Mapping of Phylogenetic Information. Bioinformatics 2003. 19(1): p. 2.
23. Cunningham, B.A., et al., Favin versus concanavalin A: Circularly permuted amino acid sequences. Proceedings of the National Academy of Sciences of the United States of America, 1979. 76(7): p. 3218-3222.
24. Peisajovich, S.G., L. Rockah, and D.S. Tawfik, Evolution of new protein topologies through multistep gene rearrangements. Nat Genet, 2006. 38(2): p. 168-74.
25. Weiner, J., 3rd and E. Bornberg-Bauer, Evolution of circular permutations in multidomain proteins. Mol Biol Evol, 2006. 23(4): p. 734-43.
26. Yu, Y. and S. Lutz, Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol, 2011. 29(1): p. 18-25.
27. Lo, W.-C., et al., CPred: a web server for predicting viable circular permutations in proteins. Nucleic Acids Research, 2012. 40(W1): p. W232-W237.
28. Hamada, H. and K. Shiraki, l-Argininamide improves the refolding more effectively than l-arginine. Journal of Biotechnology, 2007. 130(2): p. 153-160.
29. Tsumoto, K., et al., Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog, 2004. 20(5): p. 1301-8.
30. Greenfield, N.J., Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc, 2006. 1(6): p. 2876-90.
31. Kikhney, A.G. and D.I. Svergun, A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Letters, 2015. 589(19PartA): p. 2570-2577.
32. Otwinowski, Z. and W. Minor, [20] Processing of X-ray diffraction data collected in oscillation mode, in Methods in Enzymology. 1997, Academic Press. p. 307-326.
33. Winn, M.D., et al., Overview of theCCP4 suite and current developments. Acta Crystallographica Section D Biological Crystallography, 2011. 67(4): p. 235-242.
34. Adams, P.D., et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography, 2010. 66(2): p. 213-221.
35. Durowoju, I.B., et al., Differential Scanning Calorimetry - A Method for Assessing the Thermal Stability and Conformation of Protein Antigen. J Vis Exp, 2017(121).
36. Morgan, C.R. and J.R. Engen, Investigating Solution-Phase Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry, in Current Protocols in Protein Science. 2009. p. 17.6.1-17.6.17.
37. Englander, S.W., et al., Protein Folding-How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry. Annu Rev Biophys, 2016. 45: p. 135-52.
38. Putnam, C.D., et al., X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev Biophys, 2007. 40(3): p. 191-285.