Title: Elementary tetrahelical protein design for diverse oxidoreductase functions Authors: Tammer A. Farid 1,4, Goutham Kodali 1,4, Lee A. Solomon 1,4, Bruce R. Lichtenstein 1,2, Molly M. Sheehan 1, Bryan A. Fry 1, Chris Bialas 1, Nathan M. Ennist 1, Jessica A. Siedlecki 1, Zhenyu Zhao 1, Matthew A. Stetz 1, Kathleen G. Valentine 1, J. L. Ross Anderson 1,3, A. Joshua Wand 1, Bohdana M. Discher 1, Christopher C. Moser 1 and P. Leslie Dutton 1*.
Supplementary Results Label Hemes N- Cap Net Charge Phe % Tm ( C) A 0 No -16 6 37 1 38, 72 2 72 C 0 No -16 6 39 1 39, 75 D 0 No -16 6 55 E 0 No -16 9 95 F 0 No -2 6 48 (-8,+6)* 2 70 G 0 No +11 6 55 H 0 Yes -16 6 46 2 68 J 0 No -16 7 54 Supplementary Table 1: Protein cofactor capacity, helical structure melting temperatures (T m ), Percentage Phenyl Alanine of sequence, Presence of N-cap, and Net Charge: * -2 charge was calculated for entire protein, each domain has a charge of -8 and plus 6 respectively.
Supplementary Figure 1: Heme extinction coefficient determination: Calculating molar absorptivity of heme in J from experimental data. Protein with heme in 20 mm CHES, 150 mm KCl, ph 9, at varying concentrations was analyzed by ICP-OES.
Supplementary Figure 2: Heme binding titration of sequence A: Soret absorbance at 412 nm vs. heme concentration. Protein concentration is 6 nm in 20 mm CHES, 150 mm KCl at ph 9.0. Heme extinction coefficient was fixed at ε = 110,000 M -1 cm -1. K D fit value (line) for heme binding is determined to be <2 nm.
Supplementary Figure 3: Redox midpoint potential titration of proteins A (Blue diamonds), F (Green circles), and G(Black squares) with heme B. Titrations were carried out in 100 mm phosphate buffer ph 8 with 100mM KCl. Oxidation/reduction potential (E h ) was adjusted by addition of either sodium dithionite or potassium ferricyanide. The data was fit to a Nernst equation yielding two potentials for F (-224mV and -150mV) and one potential for A (-290mV) and G (-150mV).
Supplementary Figure 4: Redox midpoint potential titration of protein A (green circles) and C (blue diamonds) with one heme B added. Titrations were carried out in 100 mm phosphate buffer ph 8 with 100mM KCl. Oxidation/reduction potential (E h ) was adjusted by addition of either sodium dithionite or potassium ferricyanide. The data was fit to a Nernst equation yielding one potential for both proteins at -260mV.
Supplementary Figure 5: Heme binding titration (squares) and K D determination fit (line) of C. Absorbance at 412 nm is plotted against heme concentration in µm. Protein concentration was 3.2 µm in 20 mm CHES, 150 mm KCl at ph 9.0. Heme extinction coefficient was fixed at ε = 109,700 M -1 cm -1. K D value for heme binding is determined to be 12+/- 10 nm.
Supplementary Figure 6: Heme binding titration (squares) and K D determination fit (line) of J. Absorbance at 412 nm is plotted against heme concentration in µm. Protein concentration 2.2 µm in 20 mm CHES, 150 mm KCl at ph 9.0. Heme extinction coefficient was fixed at ε = 109,700 M -1 cm -1. K D value for heme binding is determined to be 34+/- 12 nm.
Supplementary Figure 7: Heme binding titration of G: Soret absorbance at 412-800 nm vs. heme concentration. Protein concentration 5.3 µm in 20 mm CHES, 150 mm KCl at ph 9.0. Heme extinction coefficient was fixed at ε = 109,700 M -1 cm -1. K D fit value for heme binding (line) is 365 +/- 80 nm.
1.5 A 1.5 B Absorbance (412 nm) 1.0 0.5 1.0 0.5 0 5 10 15 20 25 [Heme] (um) 5 10 15 20 25 2.0 C 2.0 D Absorbance (427nm) 1.5 1.0 0.5 1.5 1.0 0.5 0 5 10 15 20 25 30 5 10 15 20 25 30 [ZnPPIX] (um) Supplementary Figure 8: Differential binding properties of A vs. J, which has only one bishistidine binding site. Protein concentration 5.3 µm in 20 mm CHES, 150 mm KCl at ph 9.0. A: A heme binding titration. The absorbance at the Soret peak of 412 nm is plotted against cofactor concentration. B: J heme binding titration. After one equivalent is bound, the free heme absorbance increases while the bound heme absorbance stays constant. C: A Zinc protoporphyrin IX titration. The Soret peak of bound ZnPP is 427 nm. Two ZnPP bind per A. D: J ZnPP titration. With one mono-his and one bis-his site, two equivalents of ZnPP still bind.
Supplementary Figure 9: ZnPP binding titration (squares) and K D determination fit (line) of F after one heme bound. Absorbance at 427 nm after subtracting bound heme spectrum is plotted against ZnPP concentration in µm. Protein concentration 1.3 µm in 20 mm CHES, 150 mm KCl at ph 9.0. K D value for ZnPP binding is determined to be 2.9+/- 0.5 µm.
Supplementary Figure 10: ZnC (SE370) binding titration (squares) and K D determination fit (line) of C after one heme bound. Absorbance at 418 nm after subtracting bound heme spectrum is plotted against ZnC concentration in µm. Protein concentration 1.2 µm in 20 mm CHES, 150 mm KCl at ph 9.0. K D value for ZnC binding is determined to be 800+/- 100 nm.
Supplementary Figure 11: Absorption spectra of maquette C (3.6 µm) with one equivalent of heme bound after purification using PD-10 size exclusion column (blue) compared to one equivalent of ZnC ( red).
Supplementary Figure 12: Absorbance changes of ZnC (SE370) in buffer, in 20 mm potassium phosphate, 150 mm KCl, ph 8.0 (black), in control His-free maquette E (blue) and His containing I (red) at 2 µm concentration. A red shift of 9 nm and increase in the absorbance of Soret peaks are observed upon binding, together with a 5 nm shift in the Qy band around 620 nm.
Supplementary Figure 13: Absorbance spectra of Alexafluor 750 with (red) and without (black) Zn pyropheophoribide a (Znpyppa) in I maquette. By design, the estimated distance between the pigments is 20 Å.
Supplementary Figure 14: Emission of Znpyppa alone in maquette (blue) and Aleaxafluor 750 alone in maquette (purple) when excited at 450 nm. Evidence of energy transfer when maquette with both Znpyppa and Alexa750 is excited at 450 nm (see Supplementary figure 13) showing appearance of Alexafluor 750 emission and decrease in fluorescence emission of Znpyppa itself at 675 nm (red).
Supplementary Figure 15: Fluoresence excitation spectra show the energy transfer, from Znpyppa to Alexafluor 750 (red). Without Znpyppa excitation spectrum of Alexfluor750 in I is shown (blue) as a control. Emission was collected at 800nm.
Supplementary Figure 16: HPLC chromatogram of A
Supplementary Figure 17: MALDI spectrum of HPLC purified A
Supplementary Figure 18: HPLC chromatogram of B
Supplementary Figure 19: MALDI spectrum of HPLC purified B
Supplementary Figure 20: HPLC chromatogram of C
Supplementary Figure 21: MALDI spectrum of HPLC purified C
Supplementary Figure 22: HPLC chromatogram of D
Supplementary Figure 23: MALDI spectrum of HPLC purified D
Supplementary Figure 24: HPLC chromatogram of E
Supplementary Figure 25: MALDI spectrum of HPLC purified E
Supplementary Figure 26: HPLC chromatogram of F
Supplementary Figure 27: MALDI spectrum of HPLC purified F
Supplementary Figure 28: HPLC chromatogram of G
Supplementary Figure 29: MALDI spectrum of HPLC purified G
Supplementary Figure 30: HPLC chromatogram of H
Supplementary Figure 31: MALDI spectrum of HPLC purified H
Supplementary Figure 32: HPLC chromatogram of I
Supplementary Figure 33: MALDI spectrum of HPLC purified I
Supplementary Figure 34: HPLC chromatogram of J
Supplementary Figure 35: MALDI spectrum of HPLC purified J
14,565.80 1250 1000 750 500 250 0 6000 8000 10,000 12,000 14,000 16,000 18,000 20,000 22,000 Supplementary Figure 36: MALDI spectrum of HPLC purified K without Flavin couple m/z
Supplementary Figure 37: HPLC chromatogram of Flavin coupled K
Supplementary Figure 38: MALDI spectrum of HPLC purified flavin coupled K
Supplementary Figure 39: HPLC chromatogram of L
Supplementary Figure 40: MALDI spectrum of HPLC purified L
40 20 95 C! (in millidegrees) 0-20 -40 Reverse 25 C Forward 25 C -60 200 220 240 260 Wavelength(nm) Supplementary Figure 41: CD spectra of A at 25 C (Black) heated to 95 C, held for 30 minutes (Red) and then cooled to 25 C (Blue). CD experiments were performed with 20 µm protein in 20 mm CHES at ph 9 with 150 mm KCl.
Supplementary Figure 42: Reduced protein I upon stopped-flow mixing with oxygen-saturated buffer at 15C shows oxidation of the heme as observed by UV-Visible spectroscopy without intermediate states. (A) shows UV-Visible spectra from reduced (blue) to oxidized (black) with intermediate spectra (grey). (B) Decay of the reduced state of heme (black) was observed by decay of the absorbance peak at 558nm with the corresponding growth of the oxidized heme (blue).
Supplementary Figure 43: Earlier maquette variant 13, 21, 32 allows water access to the core resulting in a short-lived oxyferrous state (shoulder shown in red at 574 nm, present at 50 msec. and is stable from less than 100 msec.) between the initial reduced spectrum (blue) and the final oxidized spectrum (green). Experiment carried out in 250 mm Boric Acid, 150 mm KCl ph 9. Protein concentration 11.5 µm rapidly mixed with oxygen-saturated buffer (~1 mm) at 15 C.