1

QM/MM study of Far-red Fluorescent Protein HcRed

Qiao Sun

CCMS, AIBNThe University of Queensland

2

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

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

What organisms have been transformed?

C. elegan

bacteria

Drosophila

mammals

6

7

The advantages of red fluorescent proteins High signal-to-noise ratio; Distinct spectral properties.

N2

CA2

CB2

CG2

CD1

N2_CA2_CB2_CG2: cis or transCA2_CB2_CG2_CD1: coplanar or non-coplanar

Chromophore of RFP

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

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

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

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

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

a) b)

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

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

• 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

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

ChemShell: A modular QM/MM package

GAUSSIAN

GAMESS-UK

Chemshell

Tcl scripts

GROMOS

CHARMm26MSI

Integratedroutines:

datamanagement

geometryoptimisation

moleculardynamics

genericforce fields

QM/MMcoupling

MNDO99

MOPAC

QM codes MM codes

DL_POLY*

TURBOMOLE

CHARMM27academic

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

Build Build

‘raw’ Protein (*.pdb)

SolvateSolvate

MinimisationMinimisation

MD simulationMD simulation

SamplingSampling

OptimisingOptimising

The steps of QM/MM calculations by Chemshell

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 runEasy to set upEquilibrium geometry agrees well with DFTSlight more flexible

20

Build the system1) 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.

OH

CZ

CE1

CD1

CG2

CD2

CE2

CB2

CA2

N2

C1

N3

C2

O2

CA1CB1

CG1

OE1

OE2

CYS63-OCD3

CYS63-CACYS63-CN

CA3C

SER65-N

SER65-CB

SER65-C

SER65-C

SER65-OG

SER65

CYS63O

21

5) Solvent - sphere37.crd

a) Center the water sphere on the active siteb) 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 shellf) 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

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

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.

pKa = ΔpKa + pKModel (1)ΔpKa = ΔpKGlobalDes+ΔpKLocalDes+ΔpKSDC-HB+ΔpKBKB-HB+ΔpKChgChg (2)

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

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

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

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

• QM RegionQM(46 atoms)

• QM/MM Optimize with ChemShellTurbomole: B3LYP for QM method CHARMM FF with DL_POLY as the MM method

• MM Region - ActiveDefine shell - within 10.0 Å of chromophoreDefine water shell - within 10.0 Å of

chromophore1000~2000 active MM atoms

• MM Region - FrozenEverything else (~10,000 atom)

Methods: QM/MM Optimization with ChemShell

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

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

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).

32

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

Cis-conformations Trans-conformations

model A, B and C

0.0

9.1 ~ 12.9model B

-4.4 ~ -1.1model A

12.4 ~ 19.9model C

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

Prof Sean Smith

Prof Walter Thiel

Dr Markus Dorrer

Acknowledge: