MIB VI, 16 th – 19 th September 2013, Mariánské lázně -1- Ivo Kabelka, 1 Jakub Štěpán, 1,2 Jaroslav Koča 1,2, and Petr Kulhánek 1,2 1 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, Brno, Czech Republic 2 CEITEC – Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, Czech Republic Study of BsoBI Endonuclease By Molecular Dynamics
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -2- BsoBI Nuclease type II restriction endonuclease from Geobacillus stearothermophilus optimal cleavage at 65 ˚C recognizes short palindromatic sequence C|PyCGPuG cofactor - 2 Mg 2+ atoms 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. 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 catalytic domains monomer A monomer B DNA
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -3- Experimental study Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, fluorescent probes: acceptor and donor FRET – Fluorescence Resonance Energy Transfer efficiency is proportional to acceptor-donor distance single molecule level (diluted solution) Proposed conformational changes
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -4- Motivation TBW, main questions, only text
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -5- Molecular Dynamics - complex input structure PDB ID: 1DC1 pmemd from AMBER v12 parm99SBbsc0 + TIP3P water c(NaCl) ~ 0.15 M 298 K and 1 bar protein + DNA
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -6- Molecular Dynamics – no DNA only protein DNA removed with DNA (150 ns) no DNA
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -7- Molecular Dynamics – no DNA only protein DNA removed d1
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -8- Metadynamics TBW, theory, CV, setup
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -9- MTD - Collective Variable Evolutions
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -10- MTD - Free Energy Estimates
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -11- Principal Component Analysis no DNA MTD IndexEigenValue
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -12- Opened Enzyme salt bridges
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -13- Contradictions Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, In the apo-enzyme (no cofactor present) both distances are shorter than in the complex.
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -14- Contradictions Dikić, J. The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level. Thesis, Giessen, In the apo-enzyme (no cofactor present) both distances are shorter than in the complex. MD simulations
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -15- Donor/Acceptor Structures Alexa594 CC 19 Å
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -16- Donor/Acceptor Dynamics E153C E290C "closed""open"
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -17- Conclusions Main outcomes
MIB VI, 16 th – 19 th September 2013, Mariánské lázně -18- Acknowledgements Financial support CEITEC GACR Computational resources MetaCentrum CERIT-SC