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.
Far-red Fluorescent Protein HcRed
The University of Queensland
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.
Chromophore of RFP
N2_CA2_CB2_CG2: cis or trans
CA2_CB2_CG2_CD1: coplanar or non-coplanar
*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?
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) H-bonds near cis conformation of chromophore of protein;
b) H-bonds near trans conformation of chromophore of protein.
Daresbury Laboratory, UK
Royal Institution UK
the Max-Planck-institute for coal research, Germany
MOLPROChemShell: A modular QM/MM package
*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).
‘raw’ Protein (*.pdb)
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.
SCC-DFTB (Self-consistent charge Density-Functional Tight-Binding) is
interfaced with CHARMM in a QM/MM method.
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.
MD results b)
HcRed(monomer) with solvate (radius=30Å); Hydrogen network between the cis conformation of chromophore and its surrounding of protein.
bond length is 0.079 Å.Most of bonds are well reproduce and their errors
are less than 0.003 Å.
(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.
(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)
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.
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.
Acknowledge: energies (a.u.), and relative energies (kcal/mol) for
Prof Sean Smith
Prof Walter Thiel
Dr Markus Dorrer