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

.

Supplementary References:

52 Kuethe, J. T. et al. Development of practical syntheses of potent

non-nucleoside reverse transcriptase inhibitors. Tetrahedron 65, 5013-5023

(2009).

53 Lynch, B. M., Khan, M. A., Teo, H. C. & Pedrotti, F. Pyrazolo[3,4-b]pyridines:

Syntheses, reactions, and nuclear magnetic resonance spectra. Can. J.

Chemistry 66, 420-428 (1988).

54 Cheung, M., Boloor, A. & Stafford, J. A. Efficient and regioselective synthesis

of 2-alkyl-2H-indazoles. J. Org. Chem. 68, 4093-4095 (2003).

55 Ohashi, K., Ruan, K. H., Kulmacz, R. J., Wu, K. K. & Wang, L. H. Primary

structure of human thromboxane synthase determined from the cDNA

sequence. J. Biol. Chem. 267, 789-793 (1992).

56 Yano, J. K. et al. The structure of human microsomal cytochrome P450 3A4

determined by X-ray crystallography to 2.05-A resolution. J. Biol. Chem. 279,

38091-38094 (2004).

57 Williams, P. A. et al. Crystal structures of human cytochrome P450 3A4 bound

to metyrapone and progesterone. Science 305, 683-686 (2004).

58 Ekroos, M. & Sjogren, T. Structural basis for ligand promiscuity in

cytochrome P450 3A4. Proc. Natl. Acad. Sci. U.S.A. 103, 13682-13687

(2006).

59 Fishelovitch, D., Shaik, S., Wolfson, H. J. & Nussinov, R. How does the

reductase help to regulate the catalytic cycle of cytochrome P450 3A4 using

the conserved water channel? J. Phys. Chem. B 114, 5964-5970 (2010).

60 Fiser, A., Do, R. K. & Sali, A. Modeling of loops in protein structures. Protein

Sci. 9, 1753-1773 (2000).

61 Luthy, R., Bowie, J. U. & Eisenberg, D. Assessment of protein models with

three-dimensional profiles. Nature 356, 83-85 (1992).

62 Lovell, S. C. et al. Structure validation by calpha geometry: Phi,psi and cbeta

deviation. Proteins 50, 437-450 (2003).

63 Ryckaert, J.-P., Ciccotti, G. & Berendsen, H. J. C. Numerical integration of the

cartesian equations of motion of a system with constraints: molecular

dynamics of n-alkanes. J. Comput. Phys. 23, 327-341 (1977).

64 Wu, G., Robertson, D. H., Brooks, C. L. & Vieth, M. Detailed analysis of

grid-based molecular docking: A case study of CDOCKER—A

CHARMm-based MD docking algorithm. J. Comput. Chem. 24, 1549-1562

(2003).

65 Abel, R. et al. Contribution of explicit solvent effects to the binding affinity of

small-molecule inhibitors in blood coagulation factor serine proteases.

ChemMedChem 6, 1049-1066 (2011).

66 Zhang, L. et al. Mapping hydration dynamics around a protein surface. Proc.

Natl. Acad. Sci. U.S.A. 104, 18461-18466 (2007).

67 Cornish, V. W. et al. Site-specific incorporation of biophysical probes into

proteins. Proc. Natl. Acad. Sci. U.S.A. 91, 2910-2914 (1994).

68 Rappe, A. K., Casewit, C. J., Colwell, K. S., Goddard, W. A. & Skiff, W. M.

UFF, a full periodic table force field for molecular mechanics and molecular

dynamics simulations. J. Am. Chem. Soc. 114, 10024-10035 (1992).

69 Brooks, B. R. et al. CHARMM: A program for macromolecular energy,

minimization, and dynamics calculations. J. Comput. Chem. 4, 187-217

(1983).

70 Mayo, S. L., Olafson, B. D. & Goddard, W. A. DREIDING: a generic force

field for molecular simulations. J. Phys. Chem. 94, 8897-8909 (1990).

71 Autenrieth, F., Tajkhorshid, E., Baudry, J. & Luthey-Schulten, Z. Classical

force field parameters for the heme prosthetic group of cytochrome c. J.

Comput. Chem. 25, 1613-1622 (2004).

72 Bathelt, C. M., Zurek, J., Mulholland, A. J. & Harvey, J. N. Electronic

structure of compound i in human isoforms of cytochrome P450 from

QM/MM modeling. J. Am. Chem. Soc. 127, 12900-12908 (2005).

73 Altun, A. & Thiel, W. Combined quantum mechanical/molecular mechanical

study on the pentacoordinated ferric and ferrous cytochrome P450cam

complexes. J. Phys. Chem. B 109, 1268-1280 (2004).

74 Smirnov, A. V. et al. Photophysics and biological applications of 7-azaindole

and its analogs. J. Phys. Chem. B 101, 2758-2769 (1997).

75 Gasteiger, J. & Saller, H. Calculation of the charge distribution in conjugated

systems by a quantification of the resonance concept. Angew. Chem. Int. Ed.

24, 687-689 (1985).

76 Hsu, P. Y. & Wang, L. H. Protein engineering of thromboxane synthase:

conversion of membrane-bound to soluble form. Arch. Biochem. Biophys. 416,

38-46 (2003).

77 Wang, L. H., Tsai, A. L. & Hsu, P. Y. Substrate binding is the rate-limiting step

in thromboxane synthase catalysis. J. Biol. Chem. 276, 14737-14743 (2001).

78 Gotoh, O. Substrate recognition sites in cytochrome P450 family 2 (CYP2)

proteins inferred from comparative analyses of amino acid and coding

nucleotide sequences. J. Biol. Chem. 267, 83-90 (1992).