Transcript Slide 1

QM/MM study of
Far-red Fluorescent Protein HcRed
Qiao Sun
CCMS, AIBN
The University of Queensland
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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 subcellular localisation can be controlled using molecular
biological techniques.
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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,
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but didn’t get to test it.
What organisms have been transformed?
C. elegan
Drosophila
bacteria
mammals
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The advantages of red fluorescent proteins
 High signal-to-noise ratio;
 Distinct spectral properties.
CB2
CA2
N2
CG2
CD1
Chromophore of RFP
N2_CA2_CB2_CG2: cis or trans
CA2_CB2_CG2_CD1: coplanar or non-coplanar
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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.
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Stereo view of the chromophore and contacting residues of mKate
(trans-conformation of Ph=2.0, cis-conformation of Ph=7.0).
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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.
ΦF = 0.11 at pH 10.7
ΦF = 0.002 at pH 8.0
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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 11
of the chromophores?
Target: HcRed
X-ray structure of 2.10 Å resolution
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, cis conformation in green).
* Wilmann etc, J. Mol. Biol., 2005, 349, 223.
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a)
b)
a) H-bonds near cis conformation of chromophore of protein;
b) H-bonds near trans conformation of chromophore of protein.
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Introduction
• Goals
 Treat the complete protein rather than simplified model
 Investigate the role of the protein environment
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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.
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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
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• Primary investigators of ChemShell:
 Paul Sherwood
Daresbury Laboratory, UK
 Richard Catlow
Royal Institution UK
 Walter Thiel
the Max-Planck-institute for coal research, Germany
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ChemShell: A modular QM/MM package
Chemshell
Tcl scripts
CHARMM27
academic
Integrated
routines:
CHARMm26
MSI
GAUSSIAN
TURBOMOLE
GAMESS-UK
MOLPRO
data
management
MNDO99
geometry
optimisation
MOPAC
molecular
dynamics
GROMOS
DL_POLY*
GULP
generic
force fields
QM codes
QM/MM
coupling
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).
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The steps of QM/MM calculations by Chemshell
‘raw’ Protein
(*.pdb)
Build
Minimisation
Solvate
MD simulation
Sampling
Optimising
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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
Preparing CHARMM 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
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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 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.
CE2
OH
CZ
CD2
CE1
CG2
CD1
CB2
O2
CA2
C2
N2
N3
SER65
C1
SER65-N
CA3
CA1
CB1
C
SER65-C
O
SER65-C
N
CYS63-C
CG1
CYS63-CA
SER65-CB
SER65-OG CYS63-O
CYS63
CD3
OE2
OE1
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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 nonwater 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)
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a)
Relative Energy: 0.0 kcal/mol
b)
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.
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Table 1. Calculation of the pKa value of the Glu214 and Glu146 residues
near the chromophore of HcRed using the PROPKA method.*
pKa = ΔpKa + pKModel
ΔpKa = ΔpKGlobalDes+ΔpKLocalDes+ΔpKSDC-HB+ΔpKBKB-HB+ΔpKChgChg
*H. Li, A. D. Robertson, J. H. Jensen, Proteins-Structure Function and
Bioinformatics 2005, 61, 704.
(1)
(2)
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 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.
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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
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are less than 0.003 Å.
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
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.
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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 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)
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)
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Methods: QM/MM Optimization with ChemShell
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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)
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Choose snapshots for QM/MM calculations
4 snapshots were taken at random intervals along the 400ps QM/MM
MD trajectory for QM/MM optimizations
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 chromophore29
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.
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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.
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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).
12.4 ~ 19.9
model C
0.0
9.1 ~ 12.9
model B
model A, B and C
-4.4 ~ -1.1
model A
Cis-conformations
Trans-conformations
Figure . Relative energies (kcal/mol) for cis- and trans-conformers of HcRed:
DFT(B3LYP/)/MM results for four snapshots.
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Future work
1. The reaction pathways between cis- and transconformations of chromophore within the protein
matrix will be explored computationally.
2. The spectral properties of cis- and transconformations of chromophore.
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Acknowledge:
Prof Sean Smith
Prof Walter Thiel
Dr Markus Dorrer
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