Volume 113, Issue 6, Pages 1331-1341 (September 2017) From Gene to Function: Cell-Free Electrophysiological and Optical Analysis of Ion Pumps in Nanodiscs  Erik Henrich, Janina Sörmann, Peter Eberhardt, Oliver Peetz, Julija Mezhyrova, Nina Morgner, Klaus Fendler, Volker Dötsch, Josef Wachtveitl, Frank Bernhard, Christian Bamann  Biophysical Journal  Volume 113, Issue 6, Pages 1331-1341 (September 2017) DOI: 10.1016/j.bpj.2017.03.026 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Schematic representation of sample preparation and analysis. The workflow of sample generation and analysis is displayed with corresponding timespans. Structures built on PDB: 2MSC (in blue for MSP) and PDB: 3X3B (in purple for KR2) illustrate empty and KR2 loaded nanodiscs. Further symbols indicate the DNA template encoding for KR2 and the ribosomes of the cell-free expression system. Lipids (yellow coloring) are highlighted as single molecules in stick representation for empty disks and as a bilayer for KR2 loaded disks represented by spheres as boundaries and a colored area in between. Biophysical Journal 2017 113, 1331-1341DOI: (10.1016/j.bpj.2017.03.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Characterization of KR2 in nanodiscs. (A) Pictures of the protein in the reaction mix (i) and eluting from the affinity column (ii) are displayed and the absorption profiles of 280 nm (blue upper curve) and 530 nm (violet lower curve) of the gel filtration of purified KR2 in nanodiscs are shown. (Dashed line in black) Size-exclusion chromatogram (A280) of empty nanodiscs is shown. (B) LILBID-MS analysis of KR2 in nanodiscs with high (black/upper line) and low (red/lower line) laser intensities is given. Peaks are labeled with pictograms of the corresponding complexes, as indicated in the legend. (C) Closeup is given here of the monomer region of the mass spectrum measured at high laser intensity in (B). Pictograms as illustrated in (B) highlight the observed species. Peaks corresponding to attached lipids are indicated with black diamonds (number of diamonds corresponds to the number of attached lipid molecules). Biophysical Journal 2017 113, 1331-1341DOI: (10.1016/j.bpj.2017.03.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Absorption spectra of KR2 in nanodiscs. Shown here are the absorption spectra of KR2 in the presence of 50 mM KCl (dashed line) and 50 mM NaCl (solid line) at pH 7.4. Biophysical Journal 2017 113, 1331-1341DOI: (10.1016/j.bpj.2017.03.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Transport currents of KR2 in nanodiscs under stationary illumination recorded on the SSM system. (A) Current traces are measured in the presence of sodium (50 mM NaCl, 20 mM HEPES pH 7.4; dashed line) or of potassium (50 mM KCl, 20 mM HEPES pH 7.4; solid line). (Arrows) Start and end of the illumination is given. (B) Action spectra of KR2 show the normalized peak current densities as a function of the excitation wavelength. The symbols and bars depict the mean ± SE (n = 4 for KCl, n = 5 for NaCl), respectively. (Dashed line) Normalized absorbance spectrum of KR2 nanodiscs in 20 mM HEPES, pH 7.4, is given. (C) Shown here is the amplitude of the peak current as a function of the light intensity of an example recording. (Solid lines) Corresponding hyperbolic fits to the data are shown. 100% light intensity equals 254 mW cm−2. (D) Normalized photocurrents as a function of time at different light intensities and in the presence of sodium are given. The corresponding light intensities are indicated. 100% light intensity equals 254 mW cm−2. To see this figure in color, go online. Biophysical Journal 2017 113, 1331-1341DOI: (10.1016/j.bpj.2017.03.026) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Flash photolysis of KR2 in nanodiscs. Transient absorbance changes of KR2 at pH 7.4 after excitation at 525 nm are shown. (A) Selected time traces in 50 mM NaCl (red dashed line) and 50 mM KCl (black dashed line) and their corresponding fits (solid lines) are given. Traces are extracted from the broadband flash photolysis measurements. (B) Full broadband flash photolysis spectra (NaCl), amplitudes are color-coded: red indicates positive, green indicates zero, and blue indicates negative absorbance changes. (C) Shown here is the DAS (NaCl) of the global fit analysis using four decay time constants. Positive amplitudes indicate a decay in absorbance change; negative amplitudes indicate a rise in absorbance change. Biophysical Journal 2017 113, 1331-1341DOI: (10.1016/j.bpj.2017.03.026) Copyright © 2017 Biophysical Society Terms and Conditions