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Supporting Information

Probing membrane enhanced protein-protein interactions in the minimal redox complex of Cytochrome-P450 and P450-reductase

Mukesh Mahajan[a], Thirupathi Ravula[a], Elke Prade[a], G. M. Anantharamaiah,[b] Ayyalusamy

Ramamoorthy*[a] [a]Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055.

[b]Department of Medicine, UAB Medical Center, Birmingham, Alabama 35294.

E-mail: ramamoor@umich.edu

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

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Materials and Methods

Materials

Monobasic and dibasic components of potassium phosphate, benzphetamine and D2O were

purchased from Sigma-Aldrich (St. Louis, MO). 1,2-dimyristoyl-sn-glycero-3-phosphocholine

(DMPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). The 5-mm symmetrical

D2O-matched Shigemi NMR microtubes were purchased from Shigemi, Inc (Allison Park, PA).

Expression and purification of full-length (fl) proteins (FBD, CYP2B4 and CYP3A4).

Unlabeled or 15N labeled fl-FBD of rat CPR was purified as described previously.[1] Briefly, fl-

FBD was expressed in E. coli C41 cells in either LB medium for unlabeled protein, or M9

minimal medium for 15N labeled samples, supplemented with 5 nM FMN. Protein expression

was induced with IPTG at OD600 = 0.6-0.7 for 16 hrs at 30 °C. Post expression, cells were

harvested at 6000 x g. The cells were lysed in Tris-Acetate buffer pH 7.4 containing by 30 µg/ml

lysozyme and protease inhibitor cocktail mini tablet for 30 mins at 4 °C, followed by sonication

(with 1 sec on/off pulse) for 5 mins. The membrane fraction was separated using

ultracentrifugation at 105,000 x g for 45 mins, and further treated with 0.3% (v/v) Triton X-100

for overnight at 4 °C. The solubilized membrane proteins were purified by DEAE anion

exchange chromatography and eluted using a NaCl gradient ranging from 0.2 M to 0.5 M in

Tris-Acetate pH 7.4 containing 1 µM FMN, 0.3% (v/v) sodium cholate. Full-length CYP2B4 and

his tagged CYP3A4 was expressed and purified as described in the literature.[2-5]

Preparation of peptide based nanodiscs. 18-residue peptide (DWFKAFYDKVAEKFKEAF)

based nanodiscs were prepared with DMPC lipids in 40 mM sodium phosphate pH 7.4. The 4F

peptide was dissolved to a stock concentration of 10 mg/ml. Homogeneous suspension of

DMPC lipids were also prepared to a stock of 20 mg/ml, vortexed and sonicated. The 4F

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peptide and DMPC solutions were mixed at a 1:1.5 (w/w) ratio, and incubated for overnight at

37 °C with gentle agitation. Prepared nanodiscs were purified by size exclusion chromatography

(SEC) using a Superdex 200 Increase 300/10 GL column operated on an ÄKTA purifier (GE

Healthcare, Freiburg, Germany). The empty nanodiscs were subjected to dynamic light

scattering (DLS) measurements on a DynaPro NanoStar instrument (Wyatt Technology Corp.,

Santa Barbara, USA) at 25 °C for 10 acquisitions of 5 second each.

Reconstitution of full-length proteins in nanodiscs. Purified proteins (fl-FBD or fl-CYP2B4 or

fl-CYP3A4) at molar ratios of 1:1.2 (protein/nanodisc) were incubated in 40 mM sodium

phosphate pH 7.4 for 16 h at 25 °C with gentle agitation. Nanodiscs anchored proteins were

further purified by SEC. Eluted fractions showing absorbance at 412 nm (CYP2B4, CYP3A4) or

454 nm (FBD) were pooled and used for further analysis. In order to form a complex, fl-FBD

was added to purified CYP2B4 reconstituted in nanodiscs at a molar ratio of 1:1. DLS and SEC

measurements confirm the increase of the hydrodynamic radius after stepwise incubation with

fl-CYP450 and fl-FBD.

Titration of substrates with nanodiscs anchored fl-CYP450. Nanodisc anchored CYP450s

were titrated with substrates (benzphetamine, butylated hydroxyl toluene and testosterone).

Titrations were performed using 1-5 µM of CYP450 in 40 mM phosphate buffer at 25°C. UV-

visible spectra were monitored with a DeNovix DS-11 Spectrophotometer from 350 to 650 nm.

Acquired data were analyzed using a principal component analysis method, followed by least-

square fitting of the spectra (in SpectraLab software package) by a set of spectral standards of

pure high-spin, low-spin, and P420 species of CYP450.[6,7] The molar fraction in high-spin state

was plotted against equivalent units of substrates.

Titration of nanodiscs anchored fl-CYP450 with fl-FBD. Nanodisc anchored fl-CYP450 were

titrated in the presence and absence of saturating concentration of the substrates. Fl-FBD was

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added to both sample and reference cuvettes, in order to take into account only the spectral

changes of CYP450, as described previously.[8,9] UV-visible spectra were monitored with a

DeNovix DS-11 Spectrophotometer from 350 to 650 nm. Acquired data were analyzed using a

principal component analysis method, followed by least-square fitting of the spectra (in

SpectraLab software package) by a set of spectral standards of pure high-spin, low-spin, and

P420 species of CYP450.[7] The molar fraction in high-spin state was plotted against fl-FBD

equivalents.

NMR experiments. All NMR experiments were carried out at 298 K (25 °C) on a Bruker Avance

II 600 and 900 MHz NMR spectrometer equipped with a triple-resonance cryoprobe (1H, 15N,

13C) on a sample containing 100 µM protein and 10% D2O in 40 mM sodium phosphate pH 7.4.

Two-dimensional 1H-15N TROSY HSQC spectra were recorded with 256 t1 increments.

Chemical shift perturbations (CSPs) of 1H and 15N backbone resonances were calculated from

1H-15N TROSY HSQC spectra using the following equation:

Where ΔδN and ΔδH are the changes in the chemical shifts of 15N and 1H, respectively; α is the scaling factor and the calculated value is 0.128.[10]

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Figure S1: UV-Vis difference spectrum of CO bound fl-CYP2B4 (A) and CYP3A4 (B) reconstituted in peptide DMPC nanodiscs show absorbance maxima at 450 nm.

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Figure S2: Monitoring benzphetamine (BZ) induced spin shift of heme iron in 4F-DMPC-nanodiscs anchored fl-CYP2B4. (A) UV-Vis spectra of free fl-CYP2B4 (black) shows absorption maxima at 417 nm indicating a resting state of the heme iron of CYP450. Upon its saturation with BZ (green), an increase in the absorption at 391 nm and decrease at 417 nm indicate a shift from low to high spin states. Spectra showing the effect of the addition of fl-FBD to nanodisc-anchored fl-CYP2B4 are shown at the indicated concentration. (B) Addition of fl-FBD to BZ saturated fl-CYP2B4 shifts the spin state from 64 to 74 %. (C) Addition of fl-FBD to nanodisc-anchored fl-CYP2B4 did not change the spin state.

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Figure S3: Monitoring butylated hydroxy toluene (BHT) induced spin shift of heme iron in nanodiscs anchored fl-CYP2B4. (A) UV-Vis spectra of free fl-CYP2B4 (black) shows absorption maxima at 417 nm indicating a resting state of the heme iron of CYP450. Upon its saturation with BHT (green), an increase in the absorption at 391 nm and decrease at 417 nm indicate a shift from low to high spin states. (B) Addition of fl-FBD to BHT saturated fl-CYP2B4 shifts the spin state equilibrium to 5.5%.

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Figure S4: Monitoring testosterone induced spin shift of heme iron in 4F-DMPC-nanodiscs anchored fl-CYP3A4. 1 μM of fl-CYP3A4 was titrated with testosterone (0.1-100 μM). (A) UV-vis spectra of free fl-CYP3A4 (black) shows an absorption maximum at 417 nm indicating a resting state of the heme iron of CYP450. Upon its titration with testosterone, the increase in the absorption intensity at 391 nm and decrease in the absorption intensity at 417 nm indicate a shift from low to high spin states. (B) Change in the spin state measured from experimental results shown in (A). (C) Addition of fl-FBD to testosterone-saturated fl-CYP3A4 shifts the spin state equilibrium from 6 to 7%.

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Figure S5: Addition of benzphetamine (BZ) to fl-CYP2B4 (black) and preformed fl-CYP2B4-fl-FBD (red) complex reconstituted in 4F-DMPC nanodiscs did not alter the spin state of heme iron.

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Figure S6: (A) Amino acid sequences of CYP 2B4 and CYP 3A4 revealing only 28 % identical. (B) Superimposed X-ray crystal structures of CYP2B411 (grey) and CYP3A412 (yellow) with R.M.S.D. of ~2.0 Å shows high similarity despite the extensive variation in the amino acid residues.

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Figure S7: 1H-15N TROSY-HSQC NMR spectra of 15N-labeled fl-FBD reconstituted in 4F-DMPC nanodiscs in the absence (red) and presence (black) of fl-CYP3A4.

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Figure S8: Chemical shift perturbation (CSPs) measured from 1H-15N TROSY-HSQC NMR spectra of 15N-labeled fl-FBD reconstituted in 4F-DMPC nanodiscs induced by the presence of fl-CYP3A4. Spectra are shown in Figure S5.

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Figure S9: Changes observed for the 1H-15N TROSY-HSQC resonances of the loop residues of 15N-labeled fl-FBD reconstituted in 4F-DMPC nanodiscs (red), upon the addition of 0.5 equivalents of fl-CYP3A4 (black) and testosterone (green).

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Figure S10: Changes observed for the 1H-15N TROSY-HSQC resonances of the loop residues of 15N-labeled fl-FBD reconstituted in 4F-DMPC nanodiscs (red) upon the addition of 0.5 equivalents of fl-CYP2B4 (black) and butylated hydroxy toluene (BHT) (green).

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Figure S11: Changes observed for the 1H-15N TROSY-HSQC resonances of the loop residues of 15N-labeled fl-FBD reconstituted in 4F-DMPC nanodiscs (red) upon the addition of 0.5 equivalents of fl-CYP2B4 (black) and 1-(4-chlorophenyl)imidazole (1-CPI) (green).

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Figure S12: Changes observed for the 1H-15N TROSY-HSQC resonances of the loop residues of 15N-labeled fl-FBD reconstituted in 4F-DMPC nanodiscs (red) upon the addition of 0.5 equivalents of fl-CYP2B4 (black) and 4-(4-chlorophenyl) imidazole (4-CPI) (green).

Table S1. Spin state fractions of CYP450 heme iron reconstituted in 4F-DMPC-nanodiscs.

Sample HS (%) LS (%) CYP420 (%) CYP2B4 5 76 18 CYP2B4-Bz 62 31 7 CYP2B4-BHT 80 16 4 CYP3A4 25 73 3 CYP3A4-Testosterone 33 63 4

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