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Supporting Information
Chen et al. 10.1073/pnas.1421536112
SI Materials and Methods
The initial model of GSSoctanoylFL·hBChE was generated by
manually docking ghrelin residues 1–5, with N-methyl substituted
at the C terminus in an extended backbone conformation, into the
hBChE active site taken from the crystal structure of full-length
recombinant hBChE (Protein Data Bank ID: 3O9M). This
docking placed ghrelin’s ammonium group atop Trp82BChE and
Phe4Ghrelin close to Phe329BChE and Tyr332BChE. Force-field parameters for Ser with n-octanoylated hydroxyl (Soctanoyl) were
generated by a published procedure (1, 2).
The energy-minimized complex was neutralized with 11
chlorides, solvated with 13,050 TIP3P water molecules (3) containing 36 NaCl molecules, and energy-minimized for 100 cycles
as above followed by 100 cycles of conjugate-gradient minimization to remove close van der Waals contacts using FF12MC.
The resulting system was then heated from 0 to 300 K at a rate of
10 K/ps under constant temperature and constant volume and
finally simulated with 100 2-ns low-mass molecular dynamics
simulations (4), each of which used a unique seed number for
initial velocities, using the PMEMD module of the AMBER
11 program. All simulations used (i) a dielectric constant of 1.0;
(ii) the Berendsen coupling algorithm; (iii) a periodic boundary
condition at a constant temperature of 300 K and a constant
pressure of 1 atm with isotropic molecule-based scaling; (iv) the
Particle Mesh Ewald method to calculate long-range electrostatic interactions; (v) a time step of 1.0 fs; (vi) SHAKE bondlength constraints applied to all bonds involving the H atom;
(vii) a protocol to save the image closest to the middle of the
“primary box” to the restart and trajectory files; (viii) formatted
restart file; (ix) the FF12MC forcefield; and (x) default values of
all other inputs of the PMEMD module. A total of 1,000 trajectories saved at 100-ps intervals during the last 1-ns period of 100
simulations were subjected to a cluster analysis using the PTRAJ
module of AmberTools 13 with the average linkage algorithm (epsilon = 2.5 Å; RMS on :[email protected]*, :[email protected], :[email protected], :112–
[email protected], :[email protected], :[email protected], :[email protected], :[email protected],
:[email protected], :[email protected], :[email protected], :[email protected]; wherein residue
1 corresponds to residue 4 of the 3O9M crystal structure). This
analysis identified six clusters of GSSoctanoylFL·hBChE conformations with respective populations of 90.9, 4.1, 2.0, 1.0, 1.0, and
1.0%. The distances shown in Fig. 3 were derived from the timeaveraged conformation of the most populated cluster. The coordinates of this conformation, residue 1 of which corresponds
to residue 4 of the 3O9M crystal structure, are provided in
Dataset S1.
1. Cornell WD, et al. (1995) A second generation force field for the simulation of proteins,
nucleic acids, and organic molecules. J Am Chem Soc 117(19):5179–5197.
2. Cieplak P, Cornell WD, Bayly C, Kollman PA (1995) Application of the multimolecule
and multiconformational RESP methodology to biopolymers: Charge derivation for
DNA, RNA, and proteins. J Comput Chem 16(11):1357–1377.
3. Jorgensen WL, Chandreskhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of
simple potential functions for simulating liquid water. J Chem Phys 79(2):926–935.
4. Pang Y-P (2014) Low-mass molecular dynamics simulation: a simple and generic technique to enhance configurational sampling. Biochem Biophys Res Commun 452(3):
Movie S1. Behavior video showing the appearance of untreated 16-mo-old male BALB/c and AAV-mBChE mut vector-treated, same-aged mice. The untreated
mice (n = 23) exhibited ragged coats and bite wounds whereas the vector-treated mice (n = 29) retained clean and glossy fur. Two representative mice from
each group are shown.
Movie S1
Chen et al.
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Other Supporting Information Files
Dataset S1 (TXT)
Chen et al.
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