1. 1 QM/MM study of Far-red Fluorescent Protein HcRed Qiao Sun CCMS, AIBN The University of Queensland

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3. 3 Fluorescent proteins  Continually produced within living cells and subject to cellular targeting, partitioning, and turnover processes as with all other proteins.  These proteins are very bright and non-toxic which means that cell and tissue development can be monitored over the long term.  Importantly, fluorescent protein expression and sub- cellular localisation can be controlled using molecular biological techniques.

4. 4 Douglas Prasher Discovery and development of fluorescent proteins Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria in 1962. Martin Chalfie expressed the gen in bacteria in 1994. It worked! Roger Y. Tsien contributed to general understanding of how GFP fluoresces. Prasher cloned the GFP gen in 1992, but didn’t get to test it.

5. 5 What organisms have been transformed? C. elegan bacteria Drosophila mammals

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7. 7 The advantages of red fluorescent proteins  High signal-to-noise ratio;  Distinct spectral properties. N2_CA2_CB2_CG2: cis or trans CA2_CB2_CG2_CD1: coplanar or non-coplanar Chromophore of RFP

8. 8 *S. Pletnev, D. Shcherbo, D. M. Chudakov, N. Pletneva, E. M. Merzlyak, A. Wlodawer, Z. Dauter, V. Pletnev, J. Biol. Chem. 2008, 283, 28980. mKate * pH-induced fluorescence efficiency

9. 9 Stereo view of the chromophore and contacting residues of mKate (trans-conformation of Ph=2.0, cis-conformation of Ph=7.0 ).

10. 10 ΦF = 0.11 at pH 10.7 ΦF = 0.002 at pH 8.0 Rtms5 J. M. Battad, P. G. Wilmann, S. Olsen, E. Byres, S. C. Smith, S. G. Dove, K. N. Turcic, R. J. Devenish, J. Rossjohn, M. Prescott, J. Mol. Biol. 2007, 368, 998.

11. 11 What is the mechanism of pH induced cis-trans isomers? How the environment affect the conformations of the chromophores? Other studies show the cis-isomers possess lower energy in vacuo and in solution.

12. 12  Experiment properties*:  cis and trans conformations;  Chromophore is mobile and flexible;  cis: fluorescent properties(645nm); trans : non-fluorescent properties.  Target: HcRed X-ray structure of 2.10 Å resolution * Wilmann etc, J. Mol. Biol., 2005, 349, 223. Stereo view of the chromophore and contacting residues of HcRed (trans conformation shown in orange, cis conformation in green).

13. 13 a) b) a) H-bonds near cis conformation of chromophore of protein; b) H-bonds near trans conformation of chromophore of protein.

14. 14 Advantages choose QM/MM QM = quantum mechanics MM = molecular mechanics  Computationally less demanding;  Realistic inclusion of major environmental effect;  High-level QM treatment of active region possible;  Results amenable to qualitative interpretation. Introduction Goals  Treat the complete protein rather than simplified model  Investigate the role of the protein environment

15. 15 Different approaches to QM/MM  QM added as an extension to MM/MD force field - CHARMM/GAMESS-UK  MM environment added to a small-molecule treatment - ONION(G98,G03) - GAMESS-UK/AMBER - GAUSSIAN/AMBER(Manchester)  Modular scheme with a range of QM and MM methods - Emphasis on flexibility - e.g. Chemshell

16. 16  Paul Sherwood Daresbury Laboratory, UK  Richard Catlow Royal Institution UK  Walter Thiel the Max-Planck-institute for coal research, Germany Primary investigators of ChemShell:

17. 17 ChemShell: A modular QM/MM package GAUSSIAN GAMESS-UK Chemshell Tcl scripts GROMOS CHARMm26 MSI Integrated routines: data management geometry optimisation molecular dynamics generic force fields QM/MM coupling MNDO99 MOPAC QM codesMM codes DL_POLY* TURBOMOLE CHARMM27 academic GULP *The MD and MM modules are based on code taken from the DL_POLY package. P. Sherwood et al, J. Mol. Struct. Theochem 632, 1-28 (2003). MOLPRO

18. 18 Build ‘raw’ Protein (*.pdb) Solvate Minimisation MD simulation Sampling Optimising The steps of QM/MM calculations by Chemshell

19. 19 Preparing CHARMM Parameters - The Parameter file SCC-DFTB method for chromophore because there is no force field parameter file for the chromophore of HcRed. Preparing CHARMM Parameters – Topology file Create the Topology file chromophore of HcRed accoring to the parameters of PDB file and X-H bond parameters is according to the calculational results of SCC-DFTB method of gas phase of chromophore Why we choose SCC-DFTB method? SCC-DFTB (Self-consistent charge Density-Functional Tight-Binding) is interfaced with CHARMM in a QM/MM method.  Fast to run  Easy to set up  Equilibrium geometry agrees well with DFT  Slight more flexible

20. 20 Build the system 1) Read parameter and topolopgy files 2) Read protein PDB file 3) Read crystal waters 4) Build model:  Define the QM region: SCC-DFTB method for chromophore and some atoms of CYS 63 and SER 65  Define the centre:CA2  Use SHAKE to freeze all X-H bonds, minimize the angles and dihedral angles of all X-H bonds, because the H-positions of the raw protein are relatively distorted.

21. 21 5) Solvent - sphere37.crd a) Center the water sphere on the active site b) Delete all waters outside of 30Å sphere and which overlap ( ROX < 2.8Å) with non- water heavy atoms c) set a miscellaneous mean field potential to prevent water molecules from vapouring off d) Minimize water shell f) Run dynamics of solvation: 100ps  fix all protein atoms outside the 20 Å sphere around CA2 atom  Constrained relax protein atoms in 20 Å sphere around CA2 atom  Relaxed all the crystal and solvation water molecules Then repeat the steps from a) to f) 5-10 times 6) Run production of dynamics:500ps(300K)

22. 22 a) b) Relative Energy: 0.0 kcal/mol Relative Energy: 4.8 kcal/mol Figure 5. a) Anionic form of the chromophore with protonation state of GLU214; b) Zwitterion form of the chromophore with deprotonation state of GLU214. *The calculations are performed on the B3LYP/6-31+G* level.

23. 23 Table 1. Calculation of the pKa value of the Glu214 and Glu146 residues near the chromophore of HcRed using the PROPKA method.* *H. Li, A. D. Robertson, J. H. Jensen, Proteins-Structure Function and Bioinformatics 2005, 61, 704. pK a = ΔpK a + pK Model (1) ΔpK a = ΔpK GlobalDes +ΔpK LocalDes +ΔpK SDC-HB +ΔpK BKB-HB +ΔpK ChgChg ( 2)

24. 24  Model A (acidic conditions): Glu214 and Glu146 are protonated;  Model B (under neutral conditions): Glu146 deprotonated, Glu214 protonated;  Model C (basic conditions): Glu214 and Glu146 are deprotonated.

25. 25 MD results HcRed(monomer) with solvate (radius=30Å); Hydrogen network between the cis conformation of chromophore and its surrounding of protein.  The root-mean-square (rms) deviation between X-ray and average MD bond length is 0.079 Å. Most of bonds are well reproduce and their errors are less than 0.003 Å.

26. 26 Dihedral angle of N2_CA2_CB2_CG2: (1) X-ray 1YZW pdb = 0.0 º (2) MD average= 6.4 º (3) Deviation between (1) and (2)= 6.4 º Dihedral angle of CA2_CB2_CG2_CD1: (1) X-ray 1YZW pdb = 8.4 º (2) MD average= 6.2 º (3) Deviation between (1) and (2)= 2.2º The MD calculation of the anionic forms of the chromophore show that cis conformations of the chromophore in the protein are nearly coplanar. Histogram of dihedral angle (º) implied in the surrounding of the chromophore (chain B, cis conformation).

27. 27 Bond distance of O(CRO)_NE2(GLN107) (1) X-ray 1YZW pdb = 3.091 (Å) (2) MD average= 3.054 (Å) (3) Deviation between (1) and (2)= 0.035(Å) Bond distance of OH(CRO)_OG(SER144) (1) X-ray 1YZW pdb = 2.601 (Å) (2) MD average= 2.856 (Å) (3) Deviation between (1) and (2)= 0.255(Å) Bond distance of N2(CRO)_OE2(GLU214) (1) X-ray 1YZW pdb = 2.966 (Å) (2) MD average= 3.447 (Å) (3) Deviation between (1) and (2)= 0.481(Å ) Bond distance of O2(CRO)_NH2(ARG93) (1) X-ray 1YZW pdb = 3.190 (Å) (2) MD average= 2.676 (Å) (3) Deviation between (1) and (2)= 0.514(Å)

28. 28 QM Region  QM(46 atoms) QM/MM Optimize with ChemShell  Turbomole: B3LYP for QM method  CHARMM FF with DL_POLY as the MM method MM Region - Active  Define shell - within 10.0 Å of chromophore  Define water shell - within 10.0 Å of chromophore  1000~2000 active MM atoms MM Region - Frozen  Everything else (~10,000 atom) Methods: QM/MM Optimization with ChemShell

29. 29 a) b) The calculated structures on DFT/CHARMM level. Hydrogen network between the cis conformation of chromophore and its surrounding; b) Hydrogen network between the trans conformation of chromophore and its surrounding. Choose snapshots for QM/MM calculations 4 snapshots were taken at random intervals along the 400ps QM/MM MD trajectory for QM/MM optimizations

30. 30 Table 1. Relevant dihedral angles (º) and hydrogen bond distances (Å) for the cis- and trans-chromophore in model B of HcRed: DFT/MM optimized values for snapshots 1-4 and experimental data.

31. 31 Table 2. QM energies (a.u.), MM energies (a.u.), total QM/MM energies (a.u.), and relative energies (kcal/mol) for cis- and trans-conformers in model B of HcRed: DFT(B3LYP/SV(P))/MM results for snapshots 1-4. QM/MM energies: E total =E(QM,MM)+E(MM,QM) E(QM,MM) is the sum of E QM and the energy resulting from the electrostatic interaction between the QM and MM subsystems, E(MM,QM) is the sum of E MM and the vdW and bonded interactions between the MM and QM subsystems. Conclusions:  cis-conformations of the chromophore in the protein are coplanar.  The trans is more stable than the cis conformation by about 9.1 ~ 12.9 kcal/mol (consistent with the experimentally observed preference for the cis chromophore).

32. 32 Figure. Relative energies (kcal/mol) for cis- and trans-conformers of HcRed: DFT(B3LYP/)/MM results for four snapshots.

33. 33 Future work 1.The reaction pathways between cis- and trans- conformations of chromophore within the protein matrix will be explored computationally. 2.The spectral properties of cis- and trans- conformations of chromophore.

34. 34 Prof Sean Smith Prof Walter Thiel Dr Markus Dorrer Acknowledge: