2.8.1 Using delphi to create the electrostatic potential map
Delphi is used to calculate the electrostatic potential in and around macromolecules(133). The chromophore is not a standard amino acid, which is necessary to have one more step to cre- ate the atomic radii and charge files for the chromophore manually, which are shown below:
mc-cro.crg
! united atom amber charges for chromophore of mCherry CE CH6 0.007
N1 CH6 -0.520 CG1 CH6 0.090 CB1 CH6 0.037 SD CH6 -0.025
CA1 CH6 0.137 C1 CH6 0.526 N2 CH6 -0.277 OH CH6 -0.368 CD2 CH6 -0.035 CE2 CH6 0.100 CZ CH6 -0.121 CE1 CH6 0.100 CD1 CH6 -0.035 CG2 CH6 -0.001 CB2 CH6 0.022 CA2 CH6 0.245 C2 CH6 0.526 O2 CH6 -0.500 N3 CH6 -0.194 CA3 CH6 0.246 C3 CH6 0.526 O3 CH6 -0.500 mc-cro.siz N1 CH6 1.5 CE CH6 1.7 SD CH6 1.85 CG1 CH6 2.0
CB1 CH6 2.0 CA1 CH6 2.0 C1 CH6 1.7 N2 CH6 1.5 OH CH6 1.4 CD2 CH6 1.7 CE2 CH6 1.7 CZ CH6 1.7 CE1 CH6 1.7 CD1 CH6 1.7 CG2 CH6 1.7 CB2 CH6 2.0 CA2 CH6 1.7 C2 CH6 1.7 O2 CH6 1.4 N3 CH6 1.5 CA3 CH6 2.0 C3 CH6 1.7 O3 CH6 1.4
The parameter file (fort.10) is the one to allow Delphi to configure the library, the protein and the environment conditions. The size and charge files (For ) and the protein (pdb) are re- quired. Other parameters, like grid size and scale, salt concentration, dielectric constant, etc.,
should be defined by users. One example of the parameter file (fort.10) for running Delphi shows below:
gsize=150 #grid size
scale=2.0 #lattice spacing in number of Angstrom in(pdb,file="2h5q.pdb") #input pdb file
in(siz,file="parseres-mc.siz #input atomic radii library in(crg,file="parseres-mc.crg") #input atomic charge library indi=2.0 #dielectric constant inside
exdi=80.0 #dielectric constant outside
prbrad=1.4 #radius (in A) of the water probe mol salt=0.02 #salt concentration
bndcon=2 #2-Debue-Huckel potential of the equ. dipole maxc=0.0001 #potential max change threshold
linit=800 #linear iterations
out(modpdb,file="2h5q_delphi.pdb") # output modified pdb
out(phi,file="2h5q.phi",format=2) # output electrostatic density map energy(s,c,g) # write out energy
Users have four choices for boundary conditions (bndcon): potential zero; Debue-Huckel potential of the equivalent dipole to the molecular charge distribution; focusing from a previous phi-map calculation; Debue-Huckel potential of all charges (coulombic). The flag of MAXC is suggested to be defined by the DELPHI MANUAL, which is the convergence threshold value based on root mean square change of potential. A low maximum change in the potential is a sign
for a good convergence, for example, 0.001 [kT/e]. The iteration (linit) needs to be sufficient to allow the final maximum change less than the maxc value defined. Energy output can be written in grid (g), salvation (s), coulombic (c), analytical surface (as) and analytical grid (ag).
2.8.2 Using Modeller to build the structure model
Modeller is a tool used to build a structural model based on a given template, which is suitable for building the model of mCherry variants. Modeller is written by Python, which can be run either in the Python shell or under prompt with the script. The template is necessary for the model construct. In our case, the template is the wild type mCherry. The variants were aligned with the w.t. mCherry. The alignment file (*.ali) was generated by Modeller, which was used for building models. The scripts for alignment and model building are shown below:
from modeller import * env = environ()
aln = alignment(env)
mdl = model(env, file='mcwt', model_segment=('FIRST:A','LAST:A')) aln.append_model(mdl, align_codes='mcwt', atom_files='mcwt.pdb') aln.append(file='variant.ali', align_codes='variant')
aln.align2d()
aln.write(file='v-wt.ali', alignment_format='PIR') aln.write(file='v-wt.pap', alignment_format='PAP') The script for running Modeller is:
from modeller import *
from modeller.automodel import * env = environ()
a = automodel(env, alnfile='v-wt.ali', knowns='mcwt', sequence='variant', assess_methods=(assess.DOPE, assess.GA341)) a.starting_model = 1 a.ending_model = 5 a.make()
The number of models generated can be defined by user with the flag a.starting_model and a.ending_model. The scoring methods of DOPE and GA341 were used in this model build- ing. DOPE (discrete optimized protein energy) model score is designed for selecting the best structure by the satisfaction of global spatial restrains from a collection of models built by Mod- eller (134). DOPE is derived from an atomic distance-dependent statistical potential from a sam- ple of native structures independent on any adjustable parameters, which uses the noninteracting atoms in a homogeneous sphere as the reference state. As a result, it accounts for the finite and spherical shape of the native structure. GA341 is the scoring function based on the compactness of the model, a combined statistical potential z-score and the percentage sequence identity of the alignment (135).
The program was run under the cmd in Windows XP by typing the command: >mod9v8 aln.py
2.8.3 Using AutoDOCK-VINA to dock calcium ion to designed calcium binding pro- teins
AutoDOCK-VINA is used to predict the interaction between a ligand and a protein. VI- NA is the newest version of Autodock, which improves the performance of the old version and keep the algorithm. The format of input file of both ligand and protein should be pdbqt, which can be generated by Autodock Tools or MGL Tools (The Scripps Research Institute). The polar hydrogens should be added first and charge will be assigned and written into the pdbqt file. This input file, including the coordinates and the partial charge of each atom, will be generated auto- matically when choosing the macromolecule in the menu of Grid. The grid size and positions can be set in the Grid Box, which is required for VINA.
To run VINA, the configuration file named conf.txt should be written by the user. The input receptor (protein) and ligand pdbqt files are directed, as well as the grid information. One example as: receptor = MC_P1-min.pdbqt ligand = ca.pdbqt center_x = 40.819 center_y = 18.051 center_z = 13.987 size_x = 48 size_y = 54 size_z = 50 exhaustiveness = 8
The center_x/y/z and size_x/y/z are the parameters of the grid box, which is read from the MGL Tools. Under cmd, go to the directory of all pdbqt files, and type:
“\Program Files\The Scripps Research Institute\Vina\vina.exe” –config conf.txt –log log.txt
2.8.4 Molecular dynamic simulation of CatchER
2.8.4.1 Preparation of the chromophore using GAUSSIAN and RESP
RESP (Restrained Electrostatic Potential) charge derivation is used for assign partial charges for a “new” molecule. It is a crucial step in the molecular dynamics simulation based on AMBER and CHARMM force fields. To obtain the partial charges by this method, quantum chemistry calculation is first applied to optimize the molecule to output a stable minimized struc- ture and subsequently to calculate a molecular electrostatic potential (MEP) in a three-
dimensional grid which is generated by GAUSSIAN or GAMESS. RESP program is then used to fit an atom-centered charge to MEP.
Here, R.E.D. (RESP ESP charge Derive) program was used for derive RESP charges for the chromophore. In the R.E.D. program, two steps of the quantum chemistry calculation and the final charge fitting are integrated and no format conversion is required. This program is executed under LINUX platform. The initial input file of the chromophore in PDB format is directly ex- tracted from the mCherry structure (PDB ID: 2H5Q). Then Ante_R.E.D. program will generate five files for further quantum calculation, including P2N file (.p2n), PDB output (.pdb),
GAMESS input file (.inp), GAUSSIAN input file (.com), and the file containing atom connec- tives (.txt).
perl Ante_RED.pl cro.pdb > Ante_RED.log
GAUSSIAN03 was used for the quantum chemistry calculation to generate the input file for RESP fitting:
g03 < cro.com > cro-Gaussian.log
However, the frequency job in this the QM output file can not be recognized by the fol- lowing program, so it has to be removed before running RESP fitting (containing “Frequencies”). And the filenames of the p2N and GAUSSIAN output should be changed to Mol_red1.p2n and Mol_red1.log, respectively. Since the chromophore is the non-standard residue in the middle of the peptide, so some charge restriction should be defined, like the peptide backbone N, C, H and O. In this case, charges for N, C and H will be defined and one remark will be added into the P2N file :
REMARKS ……
Finally, the RESP charge is fit by the command: perl RED-vIII.4.pl > RED-vIII.log
2.8.4.2 Structure minimization and MD simulation by AMBER
AMBER package includes several programs for molecular simulation. In the study of mCherry variants, AMBER is used for energy minimization and simulation. AMBER requires users to prepare the pdb files for minimization. Sometimes the protein pdb files are involved in non-standard amino acids. In these cases, AMBER does not recognize it and ignores it, like the chromophore in GFP and mCherry. If we want AMBER to take account for them, the specific parameter files have to be created. Antechamber is one of the tools in AMBER package, which is
used to deal with the small organic molecules. tLeap can further generate the files of the complex of protein and ligand ready for Sander. Protein pdb file should be checked first to avoid the un- expected errors and all hydrogen should be removed at this step. The chromophore pdb file is prepared by extracting the chromophore from the mCherry directly. Antechamber is used to gen- erate the amber preparation input file in the format of prepi and the bond parameter file in the format of frcmod. tLeap is used to generate the library of the chromophore, cro.lib, which is fur- ther used for building the SANDER input files of the complex (topology and coordinates). All the scripts and commands are shown in Appendix A.2.