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Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2 , and Petr Kulhánek 1,2

Study of BsoBI Endonuclease By Molecular Dynamics. Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2 , and Petr Kulhánek 1,2 kulhanek@chemi.muni.cz 1 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

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Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2 , and Petr Kulhánek 1,2

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  1. Study of BsoBI Endonuclease By Molecular Dynamics Ivo Kabelka,1 Jakub Štěpán,1,2 Jaroslav Koča1,2, andPetr Kulhánek1,2 kulhanek@chemi.muni.cz 1National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic 2CEITEC – Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

  2. BsoBINuclease • type II restriction endonuclease from Geobacillusstearothermophilus • optimal cleavage at 65 ˚C • recognizes short palindromatic sequence C|PyCGPuG • cofactor - 2 Mg2+ atoms • homodimeric enzyme - both in free form and in complex with DNA (2x323 AA) • capable of cleaving both linear and circular DNA molecules • 20 Å long tunnel for DNA recognition • complete encirclement of DNA helical domains monomer A DNA monomer B catalytic domains Ruan, H.; Lunnen, K. D.; Scott, M. E.; Moran, L. S.; Slatko, B. E.; Pelletier, J. J.; Hess, E. J.; Benner, J., 2nd; Wilson, G. G.; Xu, S. Y. Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems. Mol. Gen. Genet. 1996, 252, 695–699.

  3. Experimental study Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, 2009. • FRET – Fluorescence Resonance Energy Transfer • efficiency is proportional to acceptor-donor distance • single molecule level (diluted solution) Proposed conformational changes fluorescent probes: acceptor and donor

  4. Motivation • TBW, main questions, only text

  5. Molecular Dynamics - complex protein + DNA • input structure PDB ID: 1DC1 • pmemd from AMBER v12 • parm99SBbsc0 + TIP3P water • c(NaCl) ~ 0.15 M • 298 K and 1 bar

  6. Molecular Dynamics – no DNA only protein DNA removed with DNA (150 ns) no DNA

  7. Molecular Dynamics – no DNA only protein DNA removed d1

  8. Metadynamics • TBW, theory, CV, setup

  9. MTD - Collective Variable Evolutions

  10. MTD - Free Energy Estimates

  11. Principal Component Analysis no DNA MTD

  12. Opened Enzyme salt bridges

  13. Contradictions Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, 2009. In the apo-enzyme (no cofactor present) both distances are shorter than in the complex.

  14. Contradictions Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, 2009. In the apo-enzyme (no cofactor present) both distances are shorter than in the complex. MD simulations

  15. Donor/Acceptor Structures Cb 19 Å Alexa594

  16. Donor/Acceptor Dynamics E153C "closed" "open" E290C

  17. Conclusions • Main outcomes

  18. Acknowledgements • Financial support • CEITEC • GACR • Computational resources • MetaCentrum • CERIT-SC

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