Supplemental Materials

Location of menaquinone and menaquinol headgroups in model membranes

Cameron Van Cleave1, Heide A. Murakami1, Nuttaporn Samart1,2, Jordan T. Koehn1, Pablo Maldonado, Jr.1, Heidi D. Kreckel1, Elana J. Cope1, Andrea Basile1, Dean C. Crick3,4 and Debbie C. Crans1,3*

1 Department of Chemistry, Colorado State University, Fort Collins, CO 805232 Department of Chemistry, Rajabhat Rajanagarindra University, Chachoengsao, Thailand3 Cell and Molecular Biology Program, Colorado State University, Fort Collins, CO 805234 Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523* Debbie C. Crans; Debbie.Crans@colostate.edu

Table of ContentsS1. Stability UV-Vis Studies 2S2. Calculation of Compression Moduli 6S3. Dynamic Light Scattering Studies of RM Samples 7S4. 1D NMR Stability Studies 8S5. 2D NMR Studies of MEN, MDL, and BEN in Reverse Micelles 11

AbbreviationsMenadione, MEN; menadiol, MDL; dipalmitoylphosphatidylcholine, DPPC; dipalmitoylphosphatidylethanolamine, DPPE; aerosol-OT, AOT; reverse micelle, RM; benzoquinone, BEN

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S1. Stability UV-Vis Studies

Figure S1. UV-Vis spectra of 0.1 mM MEN in water over 60 minutes, demonstrating the stability of MEN in an aqueous solution.

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Figure S2. Full UV-Vis spectra of 0.10 mM MEN (black), 0.10 mM MDL that was fully dissolved before analysis (red), supernatant from a 0.1 mM MDL solution when MDL had just been added to water (blue), and aqueous solution added to solid MDL at the bottom of the quartz cuvette (green). Spectra are shown at times A) t = 0 min and B) t = 60 min.

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Figure S3. UV-Vis of DTT reference (black), solid MEN with solid DTT (red), and solid MDL with solid DTT (blue) over 60 minutes. Graph A) represents time t = 0 minutes, B) is at t = 5 minutes, C) is t = 15 minutes, and D) is t = 60 minutes. Figures presented in the main manuscript truncate the y-axis to 1.5 as any measurements above 1.5 have high experimental error.

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Figure S4. Full UV-Vis spectra of a) a similar amount of MDL and DTT dissolving into water over 60 minutes Figures presented in the main manuscript truncate the y-axis to 1.5 as any measurements above 1.5 have high experimental error.

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S2. Calculation of Compression Moduli

Compression moduli were calculated using OriginPro version 9.1 from the compression isotherm average results using equation S1, where CS

-1 is the compression modulus, A is the surface area, and π is the surface pressure.

C s−1=−A ( dπ

dA) (S1)

A Savitsky-Golay smoothing function was used (2nd degree polynomial, 250 points in the window).

Figure S5. Compression moduli of mixed monolayers containing menadione and either DPPE (left) or DPPC (right). Pure phospholipid monolayers are represented by solid black curves, 50:50 phospholipid:MEN by red dashed curves, and 25:75 phospholipid:MEN by blue dotted curves. There is a possible increase in rigidity for DPPE monolayers with the addition of MEN and a possible decrease in DPPC monolayers. However, it is uncertain why MEN would affect the elasticity of the DPPC monolayer in a 25:75 MEN:DPPC mixture instead of a 50:50 mixture.

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S3. Dynamic Light Scattering of RM Samples

Table S1. Hydrodynamic diameters of RM samplesSample Hydrodynamic Diameter (nm)

w0 16 RM 12.8 ± 1.5w0 16 RM with MEN 12.6 ± 1.5w0 16 RM with 90:10 MeOH:H2O “water pool”

14.5 ± 1.6

w0 16 RM with 90:10 MeOH:H2O “water pool” and MDL

14.2 ± 1.9

w0 16 RM with 80:20 MeOH:H2O “water pool”

14.2 ± 1.6

w0 16 RM with 90:10 MeOH:H2O “water pool” and MDL

16.2 ± 2.3

w0 16 RM with 70:30 MeOH:H2O “water pool”

13.9 ± 1.4

w0 16 RM with 90:10 MeOH:H2O “water pool” and MDL

16.3 ± 2.1

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S4. 1D NMR Stability Studies

Figure S6. Stability studies of MDL in d6-benzene at time 0 (top), 1 hour (middle), and 1 day (bottom). MEN peaks are indicated by *.

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Figure S7. Stability studies of MDL in CDCl3 at time 0 (top), 1 hour (middle), and 1 day (bottom). MEN peaks are indicated by *.

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Figure S8. Stability studies of MDL in D2O at time 0 (top), 1 hour (middle), and 1 day (bottom). MEN peaks are indicated by *.

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S5. 2D NMR Studies of MEN, MDL, and BEN in Reverse Micelles

Figure S9. Proton labeling scheme of AOT.

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Figure S10. Full 1H-1H 2D NMR NOESY NMR (400 MHz) spectra of MEN (50mM) in a w0 12 RM. A standard NOESY pulse consisted of 256 transients with 16 scans in the f 1

domain using a 200 ms mixing time and a 1.5 s relaxation delay.

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Figure S11. Full 1H-1H 2D NMR ROESY NMR (400 MHz) spectra of MEN (50mM) in a w0 12 RM. A standard ROESY pulse consisted of 256 transients with 16 scans in the f 1

domain using a 200 ms mixing time and a 1.5 s relaxation delay.

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Figure S12. Full 1H-1H2D NOESY (500 MHz) spectra of menadiol (30mM) in a 70:30 MeOD:D2O AOT RM suspension at 26°C. A standard NOESY pulse consisted of 256 transients with 16 scans in the f1 domain using a 200 ms mixing time and a 1.5 s relaxation delay.

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Figure S13. Full 1H-1H2D ROESY NMR (500 MHz) spectra of menadiol and menadione (30mM) in a 70:30 MeOD:D2O AOT RM suspension at 26°C. A standard ROESYAD pulse consisted of 256 transients with 16 scans in the f1 domain using a 200 ms mixing time and a 2.0 s relaxation delay.

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Figure S14. Full 1H-1H2D NOESY (400 MHz) spectra of BEN (50mM) inside w0

12 RM at 26°C. (A standard NOESY pulse consisted of 256 transients with 16 scans in the f1 domain using a 200 ms mixing time and a 1.5 s relaxation delay.

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Figure S15. Full 1H-1H 2D ROESY NMR (400 MHz) spectra of BEN (50mM) inside w0 12 RM at 26°C. A standard ROESYAD pulse consisted of 256 transients with 16 scans in the f1 domain using a 200 ms mixing time and a 1.5 s relaxation delay.

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