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QM/MM study of Far-red Fluorescent Protein HcRed. Qiao Sun CCMS, AIBN The University of Queensland. Fluorescent proteins Continually produced within living cells and subject to cellular targeting, partitioning, and turnover processes as with all other proteins.

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QM/MM study of

Far-red Fluorescent Protein HcRed

Qiao Sun


The University of Queensland

  • 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.

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.

Douglas Prasher

Prasher cloned the GFP gen in 1992,

but didn’t get to test it.

What organisms have been transformed?

C. elegan




The advantages of red fluorescent proteins

  • High signal-to-noise ratio;

  • Distinct spectral properties.

Chromophore of RFP

N2_CA2_CB2_CG2: cis or trans

CA2_CB2_CG2_CD1: coplanar or non-coplanar

pH-induced fluorescence efficiency

mKate *

*S. Pletnev, D. Shcherbo, D. M. Chudakov, N. Pletneva, E. M. Merzlyak, A. Wlodawer, Z. Dauter, V. Pletnev, J. Biol. Chem. 2008, 283, 28980.

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


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.

ΦF = 0.11 at pH 10.7

ΦF = 0.002 at pH 8.0

Other studies show the cis-isomers possess lower energy in vacuo and in solution.

What is the mechanism of pH induced cis-trans isomers?

How the environment affect the conformations of the chromophores?

  • Target: HcRed vacuo and in solution. X-ray structure of 2.10 Åresolution

  • Experiment properties*:

  • cis and trans conformations;

  • Chromophore is mobile and flexible;

  • cis: fluorescent properties(645nm);

  • trans : non-fluorescent properties.

Stereo view of the chromophore and contacting residues of HcRed (trans conformation shown in orange, cisconformation in green).

* Wilmann etc, J. Mol. Biol., 2005, 349, 223.

a) b)

a) H-bonds near cis conformation of chromophore of protein;

b) H-bonds near trans conformation of chromophore of protein.

Introduction b)

  • Goals

  • Treat the complete protein rather than simplified model

  • Investigate the role of the protein environment

  • 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.

  • Different approaches to QM/MM b)

    • QM added as an extension to MM/MD force field


    • MM environment added to a small-molecule treatment

  • - ONION(G98,G03)


  • - GAUSSIAN/AMBER(Manchester)

    • Modular scheme with a range of QM and MM methods

  • - Emphasis on flexibility

  • - e.g. Chemshell

  • Paul Sherwood

    Daresbury Laboratory, UK

  • Richard Catlow

    Royal Institution UK

  • Walter Thiel

    the Max-Planck-institute for coal research, Germany

Chemshell a modular qm mm package

MNDO99 b)



ChemShell: A modular QM/MM package




Tcl scripts











genericforce fields


QM codes

MM codes

*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).

The steps of QM/MM calculations by Chemshell b)

‘raw’ Protein (*.pdb)




MD simulation



Preparing b)CHARMM Parameters – Topology fileCreate 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

PreparingCHARMM Parameters - The Parameter file

SCC-DFTB method for chromophore because there is no force field parameter file for the chromophore of HcRed.

  • 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

  • Build the system b)

  • 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 CYS63 and SER65

  • 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.

  • 5) Solvent - sphere37.crd b)

    • 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)

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.

Table 1. Calculation of the b)pKa value of the Glu214 and Glu146 residues near the chromophore of HcRed using the PROPKA method.*

pKa = ΔpKa + pKModel (1)

ΔpKa = ΔpKGlobalDes+ΔpKLocalDes+ΔpKSDC-HB+ΔpKBKB-HB+ΔpKChgChg (2)

*H. Li, A. D. Robertson, J. H. Jensen, Proteins-Structure Function and Bioinformatics 2005, 61, 704.

  • Model A b)(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.

MD results b)

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.

Dihedral angle of N2_CA2_CB2_CG2: b)

(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

Histogram of dihedral angle (º) implied in the surrounding of the chromophore (chain B, cis conformation).

The MD calculation of the anionic forms of the chromophore show that cis conformations of the chromophore in the protein are nearly coplanar.

Bond distance of O2(CRO)_NH2(ARG93) b)

(1) X-ray 1YZW pdb = 3.190 (Å)

(2) MD average= 2.676 (Å)

(3) Deviation between (1) and (2)= 0.514(Å)

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)

Methods qm mm optimization with chemshell
Methods: QM/MM Optimization with ChemShell b)

  • 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)

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.

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.

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: Etotal=E(QM,MM)+E(MM,QM)

E(QM,MM) is the sum of EQM and the energy resulting from the electrostatic interaction between the QM and MM subsystems, E(MM,QM) is the sum of EMM 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).

Figure . Relative energies (kcal/mol) for energies (a.u.), and relative energies (kcal/mol) for cis- and trans-conformers of HcRed:

DFT(B3LYP/)/MM results for four snapshots.

  • Future work energies (a.u.), and relative energies (kcal/mol) for

  • The reaction pathways between cis- and trans-conformations of chromophore within the protein matrix will be explored computationally.

  • The spectral properties of cis- and trans-conformations of chromophore.

Acknowledge: energies (a.u.), and relative energies (kcal/mol) for

Prof Sean Smith

Prof Walter Thiel

Dr Markus Dorrer