scheme to target compounds. (a (2,7-aza)trp. (b) … methods synthesis of compounds 2 and...
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Supplementary Figure S1 | Scheme to target compounds. (a) The synthetic routes of
(2,7-aza)Trp. (b) Synthesis of 2,7-diazaindole and its N(2)-Me. (c) Synthesis of
N(1)-Me
300 400 500 6000.0
0.2
0.4
0.6
0.8
1.0
Em
iss
ion
(arb
. u
nit
)
Abs
Ex 290 nm
Ex 320 nm
Em 335 nm
Em 370 nm
Em 410 nm
Em 530 nm
Inte
nsit
y (
arb
. u
nit
)
Wavelength (nm)
Supplementary Figure S2 | Absorption, excitation and emission spectra of
2,7-diazaindole in neutral water. The emission spectrum was obtained via excitation
at 290 nm (blue) and 320 nm (red). The excitation spectrum was acquired by
monitoring the emission at 335 nm (dark cyan), 370 nm (cyan), 410 nm (magenta)
and 530 nm (green).
250 300 350 400 450 500 550 600
0.0
0.2
0.4
0.6
0.8
1.0
Em
issio
n (
arb
. u
nit
)
2,7-diazaindole
N(1)-Me
N(2)-Me
2,7-diazaindole
Ex:320 nm
Inte
ns
ity
(a
rb.
un
it)
Wavelength (nm)
Supplementary Figure S3 | Absorption (dashed line) and emission (solid line)
spectra of 2,7-diazaindole, N(1)-Me and N(2)-Me in neutral water. The emission was
obtained via excitation at 290 nm.
300 400 500
0.0
0.2
0.4
0.6
0.8
1.0
Em
iss
ion
(arb
. u
nit
)
Ab
so
rpti
on
(arb
. u
nit
)
Wavelength (nm)
MeOH
THF
acetonitrile
benzene
Supplementary Figure S4 | Absorption (dashed line) and emission (solid line)
spectra of 2,7-diazaindole in various solvents. The emission was obtained via
excitation at 290 nm.
Supplementary Figure S5 | Comparative sequence alignment between TXAS and
representative cytochrome P450 crystal structures of P450cam, BM3, 3A4 and its
counterpart enzyme PGIS of human (h) and zebrafish (z) origins. The P450 members
were selected from a complete alignment and all gaps are presented. The regions
corresponding to the helices and sheets in P450 have been indicated. Shaded areas
indicate the positions corresponding to the substrate/ligand-binding residues. Six
structure relation sites (SRSs) (regions according to Gotoh78
) are boxed with broken
red lines.
Supplementary Figure S6 | The comparison of secondary structure elements and
orientations (with heme structure shown as stick figures) between homologous
crystallographic structures of CYP3A4 (PDB code:1TQN), BM3 (PDB code:1JME),
hPGIS (PDB code:2IAG) and modeling structure of TXAS.
Supplementary Figure S7 | MD simulations of water solvated TXAS. (a) Snapshots
of MD simulations for the explicit water solvated (2,7-aza)Trp system containing 44
water molecules. (b) Snapshots of MD simulations for the explicit water solvated
TXAS system containing 19409 water molecules. (c) The solvent-accessible surfaces
of the active site cavities calculated using an H probe under an electrostatic potential
(ESP) surface with electrophile/nucleophile (blue/red). (d) Close-up views of the
microenvironments of five labels (2,7-aza)Trp replaced tryptophans (31, 65, 133, 203,
and 446) in TXAS, in which the adjacent waters or residues within radius of 5 Å are
depicted. For clarity, (2,7-aza)Trp is colored in green stick figure with nitrogen
colored in blue. Portions of the protein are rendered as a gray ribbon and heme shown
as stick figures, in which oxygen, nitrogen, and iron are red, blue, and brown,
respectively. Note that despite the water rich environment in W31, W133, W203, and
W446, the lack of water molecules surrounding W65 is obvious. (e) Close-up views
of the hydrogen-bonding configurations for the pure water solvated (2,7-aza)Trp and
five labels (2,7-aza)Trp replaced tryptophans (31, 65, 133, 203, and 446) in TXAS.
(2,7-aza)Trp is colored in green stick figure with nitrogen colored in blue and the
hydration water with oxygen colored in red and hydrogen in white, also the
hydrogen-bonding indicated by blue dashed line.
250 300 350 400 450 500 550 6000.0
0.2
0.4
0.6
0.8
1.0
1.2
Trp31,65,133,203,446
-TXAS
(2,7-aza)Trp31,65,133,203,446
-TXAS
(2,7-aza)Trp31,133,203,446
Phe65
-TXAS
(2,7-aza)Trp31
Phe65,133,203,446
-TXAS
(2,7-aza)Trp65
Phe31,133,203,446
-TXAS
(2,7-aza)Trp133
Phe31,65,203,446
-TXAS
(2,7-aza)Trp203
Phe31,65,133,446
-TXAS
(2,7-aza)Trp446
Phe31,65,133,203
-TXAS
Ab
so
rban
ce (
arb
. u
nit
)
Wavelength (nm)
Supplementary Figure S8 | Absorption spectra of wild-type TXAS (red) and various
(2,7-aza)Trp substituted TXAS mutants in 20 mM sodium phosphate buffer, pH 7.5;
containing 10% glycerol, 0.2% cholic acid and 0.05% lubrol.
0 20 40 60 80 100 120 140 160
0.0
0.1
0.2
0.3
0.4
0.5 TXAS
Trp65
Phe31,133,203,446
-TXAS
A
26
8 m
in-1
PGH2 (M)
Supplementary Figure S9 | Steady-state kinetics of MDA formation by recombinant
soluble form TXAS and Trp65
Phe31,133,203,446
-TXAS. Assays were carried out in buffer
(300 μl of 20 mM, pH 7.5, containing 10% glycerol, 0.2% cholic acid and 0.05%
lubrol) containing 10 nM of TXAS. Reactions were initiated by addition of indicated
amounts of PGH2 at 23 °C. Reactions were followed by absorbance increase at 268
nm, and the first 15 sec of absorbance changes were used for activity determinations.
Based on the non-linear regression analysis (shown as the solid line), the Km for PGH2
and Vmax for MDA formation are 22.7 μM and 0.46 A268/min (TXAS), 22.1 μM and
0.42 A268/min (Trp65
Phe31,133,203,446
-TXAS), respectively.
Supplementary Figure S10 |The 1H-NMR spectrum of (2,7-aza)Trp in D2O at 274
K.
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
pp
m
3.2817
3.2932
3.3144
3.3198
3.3259
3.3324
3.3524
3.3651
4.1794
4.1917
4.2035
4.7000
7.1713
7.1829
7.1875
7.1991
8.2337
8.2359
8.2453
8.2475
8.4671
8.4693
8.4833
8.4855
2.041
1.007
1.002
0.999
1.000
NAME choupt-shenjy-13Jul15-d2o
EXPNO 274
PROCNO 1
Date_ 20130715
Time 17.39
INSTRUM spect
PROBHD 5 mm CPPBBO BB
PULPROG zg30
TD 32768
SOLVENT D2O
NS 512
DS 0
SWH 10000.000 Hz
FIDRES 0.305176 Hz
AQ 1.6384500 sec
RG 25.4
DW 50.000 usec
DE 25.73 usec
TE 274.0 K
D1 1.00000000 sec
TD0 1
======== CHANNEL f1 ========
SFO1 500.1730010 MHz
NUC1 1H
P1 11.30 usec
SI 65536
SF 500.1700230 MHz
WDW QSINE
SSB 2
LB 0.00 Hz
GB 0
PC 1.00
500MHz 1H D2O 274K
300 350 400 450 500 550
0.0
5.0x105
1.0x106
1.5x106
N7-H
N2-H
27.8 mM
55.6 mM
277.8 mM
555.6 mM
833.3 mM
1.1 M
1.4 M
1.7 M
1.9 M
13.9 M
24.3 M
32.1 M
38.0 M
42.4 M
45.7 M
48.1 M
50.0 M
51.4 M
Em
iss
ion
(c
ou
nt)
Wavelength (nm)
a
N1-H
300 350 400 450 500 550
0.0
5.0x105
1.0x106
1.5x106
N7-H
N2-H
N1-H
b 27.8 mM
55.6 mM
277.8 mM
555.6 mM
833.3 mM
1.1 M
1.4 M
1.7 M
1.9 M
13.9 M
24.3 M
32.1 M
38.0 M
Em
iss
ion
(c
ou
nt)
Wavelength (nm)
Supplementary Figure S11| The emission spectra of 2,7-diazaindole (5 10-5
M)
titrated with H2O in solvents. (a) In tetrahydrofuran. (b) In acetonitrile.
0 1 2 3 4 5 6 7-0.04
-0.02
0.00
0.02
0.04
Inte
nsity (
arb
. u
nit)
Time (ns)
a
0 1 2 3 4 5 6 7
-0.1
0.0
0.1
Inte
nsity (
arb
. u
nit)b
Time (ns)
0 10 20 30 40 50-0.06
-0.03
0.00
0.03
0.06
Inte
nsity (
arb
. u
nit)c
Time (ns)0 10 20 30
-0.10
-0.05
0.00
0.05
0.10
Time (ns)
Inte
nsity (
arb
. u
nit)d
0 10 20 30
-0.1
0.0
0.1
Time (ns)
Inte
nsity (
arb
. u
nit)
e
Supplementary Figure S12 | The residuals of the fit of emission decay dynamics of
(a) (2,7-aza)Trp monitored at 320 nm. (b) (2,7-aza)Trp monitored at 540 nm. (c)
(2,7-aza)Trp monitored at 380 nm. (d) N(1)-Me. (e) N(2)-Me.
Supplementary Figure S13 | Radial distribution functions. (a) The pair radial
distribution function g(r) between (2,7-aza)Trp molecule and water in the pure water
solution. (b) The pair radial distribution function between Trp residue and water in the
wild-type TXAS aqueous solution. (c) The pair radial distribution function between
(2,7-aza)Trp and water in the five Trp-sites (2,7-aza)Trp-TXAS mutant aqueous
solution. All of the g(r) functions are averaged over the total 1 ns equilibrium MD
simulation trajectory collection, respectively.
Supplementary Figure S14 | Water molecules in the coordination shell and hydration
layer. (a) Illustration of the number of molecules in the coordination shell to the
corresponding radial distribution function g(r). (b) Number of water molecules in the
thickness of the hydration layer, 5 angstroms, for (2,7-aza)Trp dissolved in the
aqueous solution and the five Trp-sites in the wild-type TXAS aqueous solution.
Number of water molecules was counted for each site in the final 100 ps of total 1 ns
equilibrium MD simulation trajectory collection, respectively.
Supplementary Methods
Synthesis of compounds 2 and (2,7-aza)Trp
Compound 2
NaH (60%, 0.22 g, 5.5 mmol) was added to a solution of diethylacetamidomalonate
(1.08 g, 5.0 mmol) dissolved in THF (30 mL) at 0 °C under N2. After 1.5 h, 152
(1.55
g , 5.0 mmol) in THF (30 mL) was added to the mixture and the solution was stirred
at RT for 2 h, followed by further reflux at 55-60 °C overnight. After cooling, the
mixture was quenched with ice and extracted with CH2Cl2. The organic layer was
dried over MgSO4, filtered and evaporated. The crude product was purified by silica
gel column chromatography with CH2Cl2/EtOAc mixture as eluent to afford 2 (0.67 g,
30%) as white solids. 1H NMR (400 MHz, CDCl3): δ 8.69 (dd, J = 4.4, 1.6 Hz, 1H),
7.93 (dd, J = 8.4, 1.6 Hz, 1H), 7.24-7.21 (m, 1H), 6.65 (s, 1H), 4.32-4.21 (m, 4H),
4.03 (s, 2H), 1.84 (s, 3H), 1.64 (s, 9H), 1.30-1.23 (m, 6H). 13
C NMR (100 MHz,
CDCl3) : δ 169.5, 166.9, 152.1, 150.5, 147.6, 144.2, 129.1, 118.9, 118.4, 84.6, 66.1,
62.8, 29.6, 27.9, 22.8, 13.8. HRMS calcd. for C21H29N4O7 (M+H)+: 449.2036; found:
449.2054.
Compound (2,7-aza)Trp
Compound 2 (0.89 g, 2.0 mmol) in 12 M HCl was heated to reflux for 8 h. The
reaction mixture was concentrated under reduced pressure to afford (2,7-aza)Trp as a
white solid in the HCl salt form. 1H NMR (400 MHz, D2O): δ 8.94 (d, J = 8.0 Hz, 1H),
8.74 (d, J = 5.6 Hz, 1H), 7.68 (m, 1H), 4.67 (t, J = 6.0 Hz, 1H), 3.84-3.82 (m, 2H).
13C NMR (100 MHz, D2O): δ 171.2, 144.1, 142.8, 142.1, 139.4, 119.0, 117.1, 52.0,
26.9. HRMS calcd. for C9H11N4O2 (M+H)+: 207.0882; found: 207.0896.
Synthesis of compounds 2,7-diazaindole and N(2)-Me
Compound 2,7-diazaindole
Compound 2,7-diazaindole was prepared according to literature method53
. A mixture
of 2-chloronicotinaldehyde (1.00 g, 7.0 mmol) and hydrazine hydrate (10 mL, 200
mmol) in ethanol (50 mL) was heated under reflux for 16 h. The reaction mixture was
the cooled and concentrated in vacuo. The crude product was purified by silica gel
column chromatography with CH2Cl2/EtOAc mixture as eluent to afford
2,7-diazaindole (0.25 g, 30%) as white solids. 1H NMR (400 MHz, CDCl3): δ 13.59
(s, 1H), 8.64 (dd, J = 4.8, 1.6 Hz, 1H), 8.13-8.11 (m, 2H), 7.15 (dd, J = 8.4, 4.8 Hz,
1H). 13
C NMR (100 MHz, CDCl3): δ 151.7, 148.6, 133.6, 130.4, 116.8, 115.2. HRMS
calcd. for C6H6N3 (M+H)+: 120.0562; found: 120.0570.
Compound N(2)-Me
Compound N(2)-Me was prepared under modified conditions of literature procedure54
.
Trimethyloxonium tetrafluoro-borate (0.60 g, 3.6 mmol) was added to a mixture of
2,7-diazaindole (0.36 g, 3.0 mmol) in EtOAc (10 mL) and stirred for 12 h under N2.
Saturated NaHCO3 solution was added to the resulting mixture and extracted with
EtOAc. The organic layer was dried over MgSO4, filtered and evaporated. The crude
product was purified by silica gel column chromatography with CH2Cl2/EtOAc
mixture as eluent to afford 5 (0.08 g, 20%) as yellow-light oil. 1H NMR (400 MHz,
CDCl3): δ 8.66 (dd, J = 4.4, 1.6 Hz, 1H), 8.00 (dd, J = 8.4, 1.6 Hz, 1H), 7.88 (s, 1H),
7.02 (dd, J = 8.4, 4.4 Hz, 1H), 4.24 (s, 3H). 13
C NMR (100 MHz, CDCl3) : δ 158.4,
151.1, 129.4, 123.3, 117.7, 114.3, 40.9. HRMS calcd. for C7H8N3 (M+H)+: 134.0718;
found: 134.0728.
Synthesis of compound N(1)-Me
Compound N(1)-Me
Compound N(1)-Me was prepared according to literature method53
. Zinc chloride
(0.30 g, 2.2 mmol) was added to a mixture of 1-methyl-1H-pyrazol-5-amine (0.49 g, 5
mmol) in ethanol (10 mL) and 12 M HCl (0.5 mL). The mixture was heated to reflux
before 1,1,3,3-tetraethoxypropane (1.10 g, 5 mmol) in ethanol (5 mL) was added.
After 1 h the reaction mixture was poured into ice-cold water, the resultant solution
was basified with sodium hydroxide and extracted with CH2Cl2 to give 6 (0.10 g,
15%), as yellow-light oil. 1H NMR (400 MHz, CDCl3) : δ 8.60 (dd, J = 5.2, 1.2 Hz,
1H), 8.33 (dd, J = 7.6, 1.2 Hz, 1H), 8.14 (s, 1H). 7.31 (dd, J = 7.6, 5.2 Hz, 1H), 4.34
(s, 3H). 13
C NMR (100 MHz, CDCl3) : δ 148.6, 147.1, 132.1, 131.5, 116.4, 116.3,
34.5. HRMS calcd. for C7H8N3 (M+H)+: 134.0718; found: 134.0732.
The N2-H/N1-H equilibrium constant. The red-edge of the absorption (> 320 nm)
has proven to be mainly from the N2-H isomer. We then assume the molar extinction
coefficient of N(2)-Me at 320 nm ( 320 5.3 103
M-1
cm-1
) to be for the N2-H isomer.
Subsequently, we very carefully weighed 2,7-diazaindole and prepared it
volumetrically in water, after which we calculated its molarity ( 0c ) and took the
absorbance. The concentration of the N2-H isomer ( 2N Hc ) is calculated by the Beers
Lambert law 320 320 2N HA lc , where A320 is the absorbance at 320 nm, and l is the
cell length (1 cm). Knowing 2N Hc , the concentration of N1-H ( 1N Hc ) can be
deduced by 1 0 2N H N Hc c c . Accordingly, the 2 1/N H N Hc c , i.e., the equilibrium
constant, was deduced to be 2.4%.
It should be noted that comparing the excitation spectrum of N2-H (for
2,7-diazaindole) and the absorption spectrum of N(2)-Me, there seems to be a slight
red shift of the absorption spectrum in the N(2)-Me compound. Therefore, we treat
2.4% as the lower limit for the N2-H population.
Quantum yield. The emission quantum yields of N2-H are as high as 0.50 and 0.51
for 2,7-diazaindole and (2,7-aza)Trp, respectively. The measurement was done by
320 nm excitation, which is mainly attributed to the absorption of N2-H, and
comparison of its emission intensity with respect to the reference compound (quinine
sulfate) with known emission yield under identical absorbance. This is also supported
by the high quantum yield of 0.32 for the synthesized N(2)-Me compound. While the
methyl rotor commonly induces radiationless deactivation, the even higher emission
quantum yield for N2-H is thus expectable.
The high quantum yield but low population of N2-H indicates the relatively low
emission yield for N1-H and N7-H emission. Due to the indistinguishable absorption
spectra between N1-H and N2-H isomers, to measure the quantum yield of N1-H and
N7-H emission, we have to take the molar extinction coefficient of N(1)-Me (3.2
104 M
-1cm
-1) and N(2)-Me (6.7 10
4 M
-1cm
-1) at the peak wavelength 295 nm of
2,7-diazaindole, and assume these to be for N1-H and N2-H isomer, respectively. As a
result, upon the 295 nm excitation and deconvolution of the multiple emission spectra,
the emission yield of the sum of N1-H and N7-H is deduced to be 0.035 0.003 for
2,7-diazaindole. Although there are no methylated compounds available for reference,
a similar result is expected for (2,7-aza)Trp. Note that this derivation is based on a
2.4% population of the N2-H isomer of 2,7-diazaindole deduced from the red-edge of
the absorption (> 320 nm). We treat the 2.4% as a lower limit for the N2-H population.
Therefore, the deduced emission quantum yield for the N1-H (N7-H) should be the
lower limit as well.
Coupled-Cluster Hamiltonian calculation. The theoretical approach used the
Second-Order Approximate Coupled-Cluster method (CC2). We introduce a
partitioning of the Hamiltonian H into a Fock operator F and a fluctuation operator U,
describing the difference between the electron-electron repulsion and the Fock
potential, H=F+U
We then proceed to the case where the system described by H0 = F + U is perturbed
by a time-dependent one electron perturbation
H=H0+Vt
n
ni
iix
xt tiVV exp (S1)
Comparative sequence alignment and homology modeling structure of TXAS.
The protein sequences of human thromboxane synthase were obtained from the NCBI
server (GI:27371226) that was deduced from the human TXAS cDNA containing 534
amino acid residues55
. In a database for sequence searching, the search for secondary
structures was performed with the Basic Local Alignment Search Tool (BLAST
Search). The sequences of 95 known isoenzymes of the P450 family were retrieved
from the Swiss-Protein Database. These enzyme P450 proteins, which contain a heme
group, catalyze oxidation reactions that are central to steroid biosynthesis in humans
and nutrient breakdown in bacteria. Crystallography and simulations support the
opening of a substrate access channel and a water-specific conserved channel by
which gating mechanisms are operative in enzymatic activity, as revealed in P450
3A4 (CYP3A4)56-59
. The P450 3A4 shows significant 34.9% residue identity and
61.1% residue similarity with TXAS. Homology model of TXAS were built using
crystal structures of human microsomal cytochrome P450 3A4 (PDB no. 1W0E, 2J0D
and 1TQN,.. etc.) as templates. The coordinates of the crystal structures of P450s
were retrieved from the Brookhaven Protein Data Bank (PDB). The main-chain
conformation of TXAS was built with the by transferring the crystal coordinates of
P450 3A4 to the aligned components of TXAS. The conserved helices and strand
framework in P450 3A4 provided the coordinates for the main-chain backbone of
TXAS. The amino acid side chains of TXAS were placed on the main-chain model,
with each side-chain conformation set to the statistical dihedral angle. The loops
between helices were then inserted as necessary. These loop conformations were
modified using Loop Refinement method60
. The structure and orientation of the heme
in TXAS were adopted directly from the X-ray coordinate of P450 3A4. Homology
model building was carried out with Accelrys Discovery Studio version 3.1
implemented on workstations and geometry optimizations were carried out using
CHARMm. The homology model of TXAS was optimized. Estimating the accuracy
of 3D protein models is essential for interpreting them. The first step in model
evaluation is to assess if the model has the correct fold. The fold of a model can be
assessed by a high sequence similarity with the closest template by conservation of
the key functional or structural residues in the target sequence. The Profiles-3D61
and
Ramachandran Plot62
programs were used to check the validity of TXAS
3-dimensional structure by measuring the compatibility of that structure with the
sequence of the protein. Comparative sequence alignment of TXAS with P450s
secondary structure elements are shown in Supplementary Figs S5 and S6 in which
the conserved fold of the secondary structure elements and the conserved SRS
regions63
are indicated. The aromatic region containing the conserved amino acid
phenylalanine residues is labeled as “Phe-cluster”. Evaluation of these comparative
models is helpful in designing mutants to test hypotheses about the protein’s function,
including the identification of active and binding sites, the modeling of substrate
specificity, test and improvement of a sequence–structure alignment, and
confirmation of a remote structural relationship. Pymol was used for visualization.
For substrate and water docking into the protein model, CDOCKER and CHARMm
were used as well (on a Silicon Graphics Iris Workstation).The stability of the
structures was tested and their dynamics in aqueous solution was investigated. MD
simulations were carried out to 107 step of MD at 298 K for 1 ns.
Construction of water solvated TXAS systems. The hydration of TXAS model
system includes a two-stage process, in which protein surface hydration is sampling
with a surface generalized explicit solvent model, while the protein site hydration
includes another stage in which a grand canonical Monte Carlo simulation of water
sampling is performed in order to obtain (assessing) a thermodynamically reasonable
hydration level. The basic strategy involves the generation of the position, orientation
and number of water molecules in the target protein’s grid site, followed by
MD-based simulated annealing and final refinement by minimization. The annealing
schedule includes multiple heating and cooling stages and is implemented as a set of
scripts for the CDOCKER package64
. During the docking process, the van der Waals
(vdW) and electrostatic interactions are described by soft-core potentials and softened
at different levels, and the softening is removed for the final minimization. The
annealing program and soft-core potentials are applied. The grid origin was located at
the center of the active site with a minimum of 20 Å, and a grid spacing of 0.5 Å was
used. No significant differences in docking accuracy were observed using a grid
spacing ranging from 0.25 to 1.0 Å. For each defined vdW or electrostatic probe, the
interactions among all protein atoms were stored at these grid points. For ligand atoms
located between grid points, a trilinear interpolation was used to approximate the
energies. A harmonic potential with force constant was applied outside the grid
boundary. The point-charge probe was used to map electrostatic interactions in the
grid so that the grid’s vdW interactions were generated. The water-protein docking
interactions are computed from grid potential and a final minimization step is applied
to each of the water’s docking poses by full potential. The minimization consists of 50
steps of steepest descent followed by up to 200 steps of the conjugate-gradient using
an energy tolerance of 0.001 kcal mol-1
. These minimized docking poses are then
clustered based on a heavy atom RMSD approach using a 1.0 Å tolerance. The final
ranking of the water’s docking poses is based on the total docking energy (including
the intramolecular energy for waters and the water-protein interactions). A
water-protein docking is considered to be successful if the RMSD between the top
ranking (lowest energy) docking poses.
To investigate variability, 10 independent runs were performed so that a statistical
analysis could be conducted. For each of these independent runs, all of the water
starting orientations (50 replicas) were chosen using a different random seed. The
observed variation of docking resulting from these independent runs allows one to
statistically differentiate the variation in the position, orientation and the number of
water molecules in a thermodynamic equilibrium. These simulations are carried out at
the annealing temperature cycle as whole, from 300 K to 500K. We then made
attempts to alter the system by translating and reorienting a water molecule, adding a
new water molecule or removing an existing water molecule. Using these techniques
and corresponding approximations, a previously reported bulk water simulation
attains the correct density and chemical potential. Simulating more complex systems
such as protein-ligand complexes using this chemical potential for water has proven to
be effectively equivalent to having involvement of all portions of the system,
including the buried site regions, in equilibrium with bulk water65,66
. Since the
systems are usually created with insufficient water and raising the water content is
often a slow process, the first 107 moves in the MCMD simulation were carried out
using a higher excess chemical potential (-5 kcal/mol) in order to increase the water
content rapidly. The simulation continued for at least 108 moves and up to a
maximum of 109 moves depending on convergence using an excess chemical
potential of -7 kcal/mol. At the end of the simulation, the last configuration with the
most frequently encountered total number of water molecules is then saved for further
use in molecular dynamics simulations of the hydration TXAS protein
(Supplementary Fig. S7). The tertiary structure of TXAS was conformed to the
characteristic fold of the P450 superfamily. Among these five Trp residues, only W65
is indicated to lie in a hydrophobic environment, the other four residues, i.e., W31,
W133, W203 and W446, are in a more polar and water accessible microenvironment,
as manifested by explicit solvent MD simulations. The four other tryptophan residues,
the most exposed NH2-terminal residue at W31, and the protein surface located at
W203 and W446 face more solvent waters, and the particularly important W133
belongs to water accessible aqueduct channel.
MD simulations of water solvated TXAS. MD simulations were performed using
the Cerius2 software
67,68. Heme and protein parameters were taken from CHARMM
69
and Deriding70
force fields. Fe and S related missing parameters were added base on
Autenrieth et al.71
and Bathelt et al.72
. The partial charges of heme atom were
calculated by a QM (B3LYP)/MM optimization at the DFT level of theory73
. The
basis sets used were an effective core potential (ECP) LANL2DZ for the resting heme
and cysteine residue atoms. The heme Mulliken charges were added to the force field.
The DFT calculations were performed in the G03 package74
. The force field
parameters for substrate analogues were taken from Dreiding70
and partial charges
were calculated using Gasteiger algorithm75
. The system was initially minimized with
a decreasing harmonic force restraint63
on TXAS and conducted in three steps. First,
only water molecules, ions and protein residues without the active shell were allowed
to move. Next, the movement was extended to the residues within 5.0 Å around the
heme and the substrate. Finally, except heme restraint, all atoms were allowed to
move freely. In each step, energy minimization was carried out by a combination of
the steepest descent method for 50,000 steps and the conjugated gradient method for
another 50,000 steps. After minimization, each system was gradually heated from 0 to
300 K over 200 ps and equilibrated at 300 K for 1 ns for further structural analyses.
To test the protein stability upon replacing tryptophan by (2,7-aza)Trp, (2,7-aza)Trp
was geometry optimized and then replaced into the wild-type TXAS using the
CHARMM-based molecular mechanics/dynamics algorithm. The stability was
monitored with a continuous energy optimization, allowing the empirical location of
the lowest energy orientation of (2,7-aza)Trp in the tryptophan cavity of TXAS.
The atomic radial distribution function. To gain more insight into the water
structure in protein TXAS during the revision, we have calculated the atomic radial
distribution functions of Trp/(2,7-aza)Trp sites with solvated TXAS in the aqueous
solutions. The results were also used for comparison with that of (2,7-aza)Trp
molecules dissolved in the pure water (Supplementary Fig. S13).
In statistical mechanics, the radial distribution function (or pair correlation
function) g(r) in a system of particles (atoms, molecules, colloids, etc.) describes how
density varies as a function of distance from a reference particle:
TN
rrrN
rrrg
T
i
N
j
1 1
24
1
(S2)
where Ni is the number of atoms of chemical type, N is the total number of atoms, and ρ
is the overall number density. The prime indicates the terms where i = j are excluded
when the chemical types are the same.
In Supplementary Fig. S13, the first peak at around 2.2 angstroms in (a) the pure
water is suggestive of the confinement of a closely-bound water contact by the first
hydration shell to the probe (2,7-aza)Trp. Comparison with (b) and (c) shows that the
magnitude of the first peak of g(r) fluctuates in the range of 1.8–2.0 angstroms, which
is dedicated to the closest confinement water to the probe Trp/(2,7-aza)Trp site. The
results also imply that water forms hydrogen bonds more tightly with the protein site
than water itself. The hydrodynamic radii show the effective interactions in the
microenviroment of the probe site around 5 angstroms. The variation of peak
magnitude and shape is dependent on the water content as well as on the various
stereospecific orientations of the water.
Noteworthy is the largely different g(r) between those sites confined in protein
and in pure water, as revealed in Supplementary Fig. S13. This provides an insight
into the dynamic scenario for the presence of microsolvation water (bio-water) in
nanoscopically confined environments of protein. In nanoscopic environments in
protein, water molecules are in direct contact with different types of interfaces of the
site, as illustrated in Fig 4c (see main text). To further examine the extent of hydrogen
bonding between microsolvation water and the protein, we also compared the number
of water molecules in the more effective radial shell (Supplementary Fig. S14). The
water molecules are bound with the probe Trp/(2,7-aza)Trp molecules in the
coordination shell and may be represented in a two-dimensional diagram, as shown in
Supplementary Fig. S14a. The thickness of the water monolayer is approximately
1.6~2.0 angstroms, and the water distance is also about 2.7~3.1 angstroms. This gives
the thickness of the bound water layer at the maximum interfacial probe site, i.e., ~5
angstroms. It can reasonably be assumed that the bound water is composed of water
molecules hydrogen-bonded to the probe site as well as coordinated to the residues in
the vicinity of the layer of the protein nanoconfined environment.
Given in Supplementary Fig. S14b is a comparison of the water content, which
shows that there are 50~60 water molecules in the 5 angstrom thick hydration shell of
pure water. In TXAS, the Trp31-site gives apparently abundant water molecules of
~40, whereas the Trp65-site gives almost no water trapped, and other site-bound water
in the thickness layer is proportionally correlated with relevant positions in the protein,
as illustrated in Fig. 4c (see main text). In particular, no direct bound water with
Trp65 residue was revealed.
Under the current scope, by means of RDF analysis from MD trajectories, we
have investigated the characteristics for various water structures in the
nanoenviroment confinement in protein, and compared them to the bulk water in
aqueous solution. So far as we know, the water structure in protein TXAS could be
divided into different types of water: that trapped in the active site, that in channels,
that bound to the interface, and relatively unperturbed, bulk-like water. We also
demonstrated that the properties and dynamics of bio-water could not be extrapolated
from those of bulk water and need to be examined independently and compared to the
dynamical properties of bulk water.
Growth and induction of bacteria Recombinant human TXAS and its mutants
were expressed and purified as described by Hsu and Wang76
with slight
modifications. In brief, the constructs were designed by replacing the first 28 amino
acid residues in N-terminal transmembrane domain with the hydrophilic sequence,
MAKKTSS, for TXAS cDNA. A four-histidine tag was added to the C-terminus of
TXAS constructs to facilitate protein purification. The residues of a hydrophobic
patch in the putative TXAS F-G loop region (residues 222-231, IPRPILVLLL) were
individually mutated to hydrophilic residues to further optimize purification yield.
Four single mutants (I222T, V228T, L230D and L231R) revealed good solubility, but
the quadruple mutant (I222T/V228T/L230D/L231R) exhibited even better solubility
and stability, which was thus designated as a soluble-form of TXAS. The expression
constructs for the recombinant TXAS protein with single or multiple Trp mutations
were derived from above recombinant soluble-form TXAS constructs by site-directed
mutagenesis and by sub-cloning using appropriate restriction endonucleases. All
constructs were validated by DNA sequencing prior to protein expression. They were
then transformed into a tryptophan auxotrophic E. coli strain ATCC23231 for proteins
expression. Then 3 mL of the overnight culture of transformed ATCC23231 were
added into 1 L of M9 minimal medium supplemented with 2 mM MgSO4, 0.4%
Glucose, 0.1 M CaCl2, 100 μg/mL ampicillin, and 1 mM L-Trp. Growth was
facilitated with 200 rpm of shaking at 37 °C until OD600 reached 0.5; the cells were
then collected by centrifugation and re-suspended in 1 L M9 medium supplemented as
above but without L-Trp. The culture was then incubated at 30 °C (30 min, 200 rpm),
followed by the addition of 0.25 mM -ALA. After further incubation (30 °C, 15 min),
2 mM (2,7-aza)Trp and lactose (40 g) were added and the culture continued to
incubate at 30 °C (18-20 h, 180 rpm) before harvesting by centrifugation. To purify
recombinant TXAS and mutants, the cell pellets were first thawed then re-suspended
in sodium phosphate buffer (50 mM, pH 7.5 containing 0.1 M NaCl, 10% glycerol, 10
g/mL DNase and 2 mM MgCl2) at a buffer solution to cell pellet ratio of 4:1 (v:w)
and then lysed by sonication at 4 °C. TXAS proteins were further solubilized by the
addition of NP-40 (3%) with stirring (4 °C, overnight). The TXAS containing
supernatant was collected by centrifugation (4 °C, 30000 rpm) and then subject to
purification by Ni-NTA column (Qiagen), followed by hydroxyapatite
chromatography column to maximize the purity as previously described76
. The
concentration and purity of the resulting TXAS enzymes were determined by
A418/A280 ratio and adjusted to 40M by dilution with specific native buffer (20 mM
sodium phosphate, pH 7.5; containing 10% glycerol, 0.2% cholic acid and 0.05%
lubrol) to avoid protein aggregation and then stored at -80 °C. The buffer solution
exhibits a weak fluorescence at ~385 nm with lifetime ~6.3 ns, which consistently
appears in the controlled experiments, even after thorough purification. Note that the
series (2,7-aza)Trp-TXAS emission has been subtracted out in both steady state and
time-resolved approach. For the denature experiment,
(2,7-aza)Trp31,65,133,203,446
-TXAS was refluxed in a solution containing 6 M
Guanidinium chloride (GuHCl), 0.1% Sodium dodecyl sulfate (SDS) and 10%
β-Mercaptoethanol (β-Me) for 10 min.
In this study, we also measured the recombinant soluble-form TXAS activity by
monitoring the absorption of product MDA at 268 nm (268 = 31.5 mM-1
cm-1
)77
after
adding PGH2 to the native buffer. The initial rates were calculated from the slope of
the first 5th
- 20th
s of the reaction. Nonlinear least-square analysis of TXAS activity
versus PGH2 concentrations (1.5 to 150 μM), as shown in Supplementary Fig. S9,
gave the recombinant TXAS equivalent to a turnover number, kcat, of 1458 min-1
. The
purified recombinant soluble-form TXAS had about 74% catalytic activity as
compared to that of wild-type TXAS (kcat =1960min-1
)77
.
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