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Single Protein Tracking Reveals that NADPH Mediates the Insertion of Cytochrome P450-Reductase into a Biomimetic of the Endoplasmic Reticulum Carlo Barnaba, Michael J. Martinez, Evan Taylor, Adam O. Barden, and James A. Brozik J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00663 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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Journal of the American Chemical Society

Single Protein Tracking Reveals that NADPH Mediates the Insertion of Cytochrome P450-Reductase into a Biomimetic of the Endoplasmic Reticulum

Carlo Barnaba, Michael J. Martinez, Evan Taylor, Adam O. Barden, and James A. Brozik*

Washington State University, Department of Chemistry, PO Box 644630, Pullman, WA, USA, 99164-4630

*Corresponding author Dr. James A Brozik Washington State University Department of Chemistry PO Box 644630 Pullman WA, 99164-4630 Email: [email protected]

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ABSTRACT: Cytochrome P450-Reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature dependent Fluorescence Recovery After Photobleaching (FRAP). The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramatic changes in diffusion coefficient and the degree of partitioning of CPR as a function of NADPH concentration.


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1. Introduction Cytochrome P450-Reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. This redox activity allows cytochrome P450 to perform the oxidative transformations of several drugs and xenobiotics, as well as the synthesis of hormone steroids.1-2 CPR binds NADPH, which can reduce the enzyme by transferring electrons to both the FAD and FMN domains within the protein.3-4 The NADPH/FAD domain is connected to the FMN domain by a hinge region that closes when NADPH binds to CPR and transfers two electrons through a hydride ion transfer, followed by interflavin electron transfer and then opens up as the enzyme releases NADP+.5-6 CPR as well as cytochrome P450 are found in the cytoplasmic side of the endoplasmic reticulum of eukaryotic cells, and they both possess a hydrophobic N-terminal ER membrane binding domain.7-8 One of the striking characteristics of the P450 kinetic machinery is the physiological nonstoichiometric ratio between CPR and the monooxygenase component. In particular, early estimation by Estabrook reported about 20-fold excess of P450 over CPR,9 whereas more recently Reed et al. found a 5:1 P450:CPR ratio when immunochemical techniques were used.10 Since the electron transfer occurs through a 1:1 interaction between the two proteins, it still remains a conundrum how a single CPR can serve several P450s. Hypotheses have arisen over the years, including P450 quaternary organization in functional “clusters” or oligomers served by a single CPR,11-12 as well as P450 dimers with increased affinity for CPR.13 It is important to point out that, with a few exceptions,14 most of the experimental evidence has been obtained through biophysical experiments performed in model systems which are not representative of the ER environment. More advanced tools such as Nanodiscs® have shown that lipid composition can modulate the redox potential of the flavins in CPR;15 however, they ignore heterogeneous



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membrane effects and mass transport properties, which are certainly an important part of proteinprotein interactions. In this work, we supply an alternative approach to study CPR-lipid interactions. Described here is the incorporation of CPR into an ER biomimetic membrane and the direct observation of CPR mobility using single-molecule fluorescence microscopy. The ER biomimetic is fully characterized in terms of the lateral diffusivity of lipids using Fluorescence Recovery After Photobleaching (FRAP). When CYP2C9 is reconstituted with CPR into detergent free ER biomimetic membranes, high catalytic activity for the oxidation of diclofenac was observed. Most importantly, we will present the results of a series of single protein tracking studies that directly measure the mobility of CPR in the biomimetic ER as a function of its redox state. These results show two distinct protein-lipid states associated with CPR in the ER membrane. These observations may help account for the versatility of CPR as an electrontransfer catalyst for cytochrome P450.

2. Materials and Methods 2.1 Materials D-glucose, catalase, glucose oxidase, phenacetin, and diclofenac were purchased from Sigma Aldrich (St. Luis, MO). NADPH was supplied by Tocris Bioscience (Bristol, UK). 4hydroxy-diclofenac was supplied by Cayman Chemical (Ann Arbor, MI). All the lipids were purchased from Avanti Polar Lipids (Alabaster, AL).


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2.2 P450-Reductase Expression, Purification and Labeling. Full-length CPR was expressed and purified as previously reported.16 The purified protein was then dialyzed overnight at 4 oC against 100 mM phosphate buffer (pH 7.4) and 20% glycerol (v/v) using a 15 kDa MEDI tube-O-dialyzer (G-Biosciences, St. Louis, MO) and stored at -80 oC. CPR was labeled with ATTO532 Maleimide according to the protocol described by the supplier (ATTO-TEC GmbH, Siegen, Germany), with slight modifications. ATTO532 was dissolved in 100% HPLC grade acetonitrile, stored at -20°C in the dark and used in the following 24h. CPR was diluted to 15 µM in 100 mM HEPES (pH 7.4) buffer containing 150 mM NaCl and 20% glycerol (v/v). The solution was transferred in a sealed glass vial and gently degassed for 5 min under Ar while kept on ice. The dye was added to the degassed protein-containing solution in a concentration ratio 1:1.2 protein:dye, and let react under gentle stirring at 4° C overnight protected from light. The reaction was stopped by addition of a 1 M solution of DTT to a final concentration of 3 mM. The labeled CPR was purified from the excess dye by using a desalting Micro Bio-Spin® column packed with Bio-Gel® P-6 (BioRad, Hercules, CA). The gel in the column is suspended in Tris buffer (pH 7.4), but it was exchanged to 100 mM HEPES (pH 7.4) containing 150 mM NaCl according to the procedure described by the supplier. The degree of labeling was calculated according to the protocol, and was estimated to be 0.5. The labeled CPR was aliquoted, flash frozen and stored at -80 °C.

2.3 Cytochrome P450 2C9 Expression and Purification CYP2C9 was expressed in E. coli and purified as described previously.17 The purified protein was dialyzed overnight as described in the previous section for CPR. The purity of



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CYP2C9 was >95% as determined by SDS−PAGE analysis and CO bound UV/Vis absorption. Protein concentration was determined by absorbance at 450 nm using an extinction coefficient of 91 mM−1cm−1 for the CO bound protein.18

2.4 Liposomes Preparation The phospholipidic mixture used to create the ER membrane was designed according to the composition of the human endoplasmic reticulum as reported by several authors.14, 19-24 A polyethylene glycol (PEG) cushion was also incorporated into the membrane in order to increase the separation of the bilayer from the glass support and prevent unwanted interactions between CPR and the underlying substrate. Small unilamellar vesicles (SUVs) were prepared from lipid cakes made by evaporating a 1 mL chloroform solution that contained 1.42 µmol of 1,2dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1.42 µmol of 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), 1.0 µmol of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 0.35 µmol of 1,2-dilauroyl-sn-glycero-3-phospho-L-serine (sodium salt) (DLPS), 0.20 µmol of cholesterol, 0.35 µmol of L-α-phosphatidylinositol-4,5-bisphosphate, 0.19 µmol of sphingomyelin and 0.07 µmol of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethyleneglycol)-2000](ammonium salt) (PEG-PE). The concentration of PEG-PE was 1.4 mole percent. At this concentration, the PEG is in an intermediate phase between its brush and mushroom phases. Cremer and co-worker have shown that this is the optimal condition that minimizes interactions with the underlying substrate and maximizes protein diffusion within the membrane.25-26 For the cushioned assemblies with labeled lipid components used in FRAP experiments, lipid cakes also contained 0.005 µmol of 1,2-dimyristoyl-sn-glycero3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt) (Rhodamine-


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DMPE). After drying, LMVs (large multilamellar vesicles) were formed by hydrating the lipid in 1 mL of 100 mM HEPES buffer (pH 7.4) containing 5 mM CaCl2, and 140 mM NaCl (named “HEPES buffer” in the rest of the paper). The suspension of LMVs was placed in a bath sonicator at 60 ºC for 30 minutes upon which the turbid solution became translucent, indicating the formation of SUVs. The solution containing the SUVs was centrifuged for 30 minutes at 100,000 × g and the supernatant (containing the SUVs) was transferred to a 1 mL Eppendorf tube and used the same day or immediately frozen in liquid N2 and stored at -80 ºC. Liposomes composed of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), and 1,2-dilauroyl-sn-glycero-3-phospho-L-serine (DLPS) with a high concentration of detergent such as sodium cholate are widely used in CPR/P450 metabolism studies.27-28 For control experiments, liposomes of 1:1:1 by weight DLPC:DOPC:DLPS without detergent were prepared in the same fashion as the ER liposomes.

2.5 Formation of Planar Supported Lipid Bilayers Lipid bilayers were prepared according to a protocol previously described by our group.29-30 Briefly, 25 mm round borosilicate glass coverslips were first hydrophilically treated in a solution of water, concentrated nitric acid, and 30% hydrogen peroxide (1:1:1 by volume) at 80 ºC for 45 minutes, with gentle agitation to separate the coverslips. The coverslips were then rinsed with a copious amount of purified water and dried under a gentle stream of pre-purified nitrogen. A single coverslip was then placed onto a sample holder and fitted with a parafilm® gasket containing an 8-mm hole cut into its center. A 50 µL aliquot of the SUV solution was then placed in the center hole and allowed to incubate at room temperature for 40 minutes during which the SUVs fuse to the glass substrate, rupture, and form a continuous bilayer. After



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incubation, the SUV solution was carefully removed and gently rinsed 6 times with HEPES buffer.

2.6 Incorporation of CPR into Lipid Bilayers CPR was incorporated into the lipid bilayers described above by first removing (by pipette) the buffer above the bilayer and replacing it with 50 µL of a 450 pM solution of CPR in HEPES buffer and allowing the protein to self-insert into the bilayer by incubating it for 30 minutes at room temperature. After incubation, the solution was carefully removed and the membrane with the protein incorporated into it was gently washed 6 times with HEPES buffer to remove any unincorporated enzyme. For single molecule imaging experiments, an imaging buffer that contained an enzymatic oxygen scavenging system was placed on top of the sample.31 The imaging buffer contained 0.8% w/v D-glucose, 1 mg/mL glucose oxidase, and 0.04 mg/mL catalase, in 100 mM HEPES (pH 7.4), 5 mM CaCl2 and 140 mM NaCl. Measurements were made immediately after sample preparation.

2.7 Activity Assay for CPR and Cytochrome P450 2C9 Incorporated in ER Liposomes and Bilayer. Activity of CPR / CYP2C9 was measured in liposomes and planar supported lipid bilayers after protein reconstitution. For liposomes, 50 μl of ER SUV were mixed with 50 pmol purified CYP2C9, 100 pmol purified CPR and diclofenac32 at several concentrations (0, 2.5, 5, 25 and 100 µM) in HEPES buffer. The protein was incubated 30 min at room temperature and then 1 mM of NADPH was added to start the reaction. The total volume of the reaction mixture


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was 400 μL. The reaction was quenched after 10 min with a solution of acetonitrile containing a known concentration of phenacetin as an internal standard, followed by centrifugation at 10,000 × g for 10 min. Supported lipid bilayers were prepared as described previously. After washing the lipids with HEPES buffer, a solution containing 300 pM CYP2C9 and 600 pM unlabeled CPR was added to the top of the ER biomimetic and allowed to incubate for 30 min at room temperature. The excess protein was then removed as described previously, and replaced with a solution containing 100 μM diclofenac and 1 mM NADPH. The reaction was allowed to proceed for 15 min and a 40 μL aliquot was removed from the top of the sample and was added to a 10 μL quenching solution of acetonitrile containing a known concentration of phenacetin as internal standard. The aliquot was centrifuged at 10,000 × g for 10 min.

2.8 Liquid Chromatography-Mass Spectrometry Conditions for the Quantification of Metabolites. For the activity assay, an LC-20AD series high-performance liquid chromatography system (Shimadzu, Columbia, MD) fitted with a HTC PAL autosampler (LEAP Technologies, Carrboro, NC) was used to perform chromatography on a Luna® reverse-phase column (50 × 2.0 mm, 5 µm; Phenomenex, Torrance, CA), using a flow rate of 500 µL/min. Mobile phase A consisted of 0.05% formic acid and 0.2% acetic acid in water, and mobile phase B comprised 90% acetonitrile, 9.9% water, and 0.1% formic acid. Chromatographic separation was performed using a linear gradient over the next 2.2 minutes to 25% mobile phase A. Mobile phase A was then held constant at 25% over 0.5 minutes, followed by a linear gradient back to 95% A over



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1.0 minutes. Finally, the column was re-equilibrated to the initial conditions over the last 2 minutes. The total chromatographic assay time was 5.0 minutes per sample, and the retention times for internal standard (phenacetin) and metabolite (4-hydroxy-diclofenac) were 3.2 and 2.4 min, respectively. The quantification of the metabolite was conducted using an API 4000 Q-Trap tandem mass spectrometry system manufactured by Applied Biosystems/MDS Sciex (Foster City, CA) using turbospray ESI operating in positive ion mode. The optimized mass spectrometer tune parameters were as follows: collision gas, 20 psig; curtain gas, 20 psig; ion source gas 1, 60 psig; ion source gas 2, 40 psig; ion spray voltage, 5500 V; desolvation temperature, 500 ºC; declustering potential, 55 V; entrance potential, 10 V; collision energy, 30 V; collision cell exit potential, 15 V. The analyte (4-hydroxy-diclofenac) and the internal standard (phenacetin) were detected using multiple reactions monitoring mode by monitoring the m/z transition from 312.0 to 231.1 and 180.2 to 110.1, respectively. Quantitation of product was achieved by interpolating from a standard curve ranging from a 25 to 2000 nM concentration of authentic 4-hydroxy-diclofenac.

2.9 FRAP Measurements Fluorescence recovery after photobleaching (FRAP) is a robust technique for measuring the mobility and 2-D diffusion constants of fluorescently labeled membrane components in lipid bilayers. In this experiment, a small well-defined area of fluorescently labeled lipids was photobleached by a high intensity laser and its recovery was measured as a function of time. FRAP curves were collected with an IX71 fluorescence microscope equipped with a 1.0 N/A apochromatic 40x microscope objective. The fluorescence recovery was monitored with a Hg:Xe arc lamp that was passed through an optical shutter, a 555 nm bandpass filter (25 nm


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FWHM; Chroma Technologies Corp), and directed to the microscope objective with a dichroic mirror (U-N86016; Chroma Technologies Corp). A Nd:YAG laser (Power Technologies Inc.) was used to bleach a small area in the field of view. The laser was passed through a fast optical shutter (model LASSHU_PSFIB; Olympus Inc.) and directed into the microscope objective with a second dichroic mirror (QS4SLP; Chroma Technologies Corp.). The laser was attenuated such that the initial bleached area was ~70% of the original fluorescent intensity and Gaussian in shape (FWHM = 3.3 µm and Power Density = 0.11 mW / µm2). The fluorescence was collected by the objective, passed through both dichroic mirrors, then through a 605 nm bandpass filter (40 nm FWHM; Chroma Technologies Corp) and imaged onto a Hamamatsu ORCA II CCD camera. The optical shutters were synchronized and data acquisition was achieved with a custom script written within the Advanced Metamorph software suite (Olympus Inc.). Temperature control was achieved with a custom-made sample cell and objective collar in order to match and maintain the temperature at the sample and at the microscope objective. In this experimental design, two identical PID temperature controllers were utilized (Model SYL-1512A2; Auber Instruments Inc) and the temperature was monitored with matching Pt temperature sensors (Model TH100PT; Thorlabs Inc.). Data analysis was performed using a combination of the Advanced Metamorph software suite (Olympus, Inc.), IGOR 6.37 pro, and MATLAB (Mathworks Inc.). To calculate the lateral diffusion coefficients associated with the recovery of the labeled lipids equation 1 was utilized 33:

=

(1)

½



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where ω is the FWHM of the Gaussian profile of the photobleached area generated immediately after exposure to the FRAP laser, t½ is the time required for the photobleached spot to recover to ½ its maximum value, and γD is a correction factor that depends on the bleach time and the shape of the bleach area. The value of γD was 1 for our experiments. It was noted that the use of Metamorph Image Analysis Software (Olympus Inc.) in false color mode lead to artificially enhanced recoveries. Therefore, raw ‘gray scale’ data was used to determine the percent recoveries. 2.10 Single Protein Tracking. The insertion of CPR into the ER biomimetic is described above. The lateral diffusion of single CPR proteins was measured with a custom-made single molecule fluorescence microscope and experiments were carried out at 37 oC. The lateral diffusion was also measured as a function of NADPH concentration (0 M, 450 pM, and 2.25 µM). Excitation of the sample was achieved with a stabilized cw-Nd:YAG laser producing a 532 nm beam. The beam was first passed through a laser line filter (LL01-532; Semrock, Inc.), then a ¼ waveplate (WPQ05M-532; Thorlabs, Inc.) to produce a circular polarized laser beam. The beam was focused with a 150 mm achromatic lens and directed to the far edge of a 1.45 N/A apochromatic TIRF microscope objective (Olympus Inc.) with a dichroic mirror (FF545/650-Di01; Semrock, Inc.) to produce an evanescent field at the interface between the glass coverslip and the lipid bilayer (total internal reflection; the laser power before TIR was 1.1 mW). The fluorescence from single fluorescently labeled CPR was collected by the microscope objective passed through the dichroic mirror and passed through a longpass filter (HQ550LP; Chroma Technologies Corp) and imaged onto an EMCCD camera (iXon 888; Andor Tech.) with a 300 mm achromatic lens. The exposure time was set to 25 ms and the frame rate was only slightly higher at 25.02 ms. Temperature control


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was maintained at the sample and the microscope objective with a custom-made sample holder and objective collar. The sample holder and objective collar were both fitted with peltiers (TEC3-2.5; Thorlabs Inc.) and interfaced to separate Meerstetter Engineering temperature controllers (model TEC-1091). The temperature was monitored at the sample with a Pt temperature sensor (TH100PT; Thorlabs Inc.) and the hot side of the peltiers with a thermistor (TH10K; Thorlabs Inc.). Single molecule tracking was performed with an automated tracking algorithm based on the work by Crocker and Grier34 and programmed into MATLAB (The Mathworks Inc.) using modified scripts written by M. Kilfoil and co-workers at the University of Massachussets Amherst, as well as by the authors.35 The diffusion coefficients of the proteins in the biomimetic ER were determined by analysis of the mean-square displacement (MSD)36 of individually tracked membrane proteins. Individual squared displacements are given by:36 (∆ ∆ ) = (∆∆ ) + (∆∆ )

(2)

Where n is the frame number and ∆t is the time between adjacent frames (25 ms in the current study) within a particular track and ∆∆ and ∆∆ are the spatial displacement in the x and y directions for time lag n∆t. The mean-square displacement (MSD) is the average of all steps corresponding to a single time lag n∆t within the track measured for an individual protein: 〈∆ ∆ 〉 =

∑ ∆ ,∆

(3)

Where N is the total number of steps corresponding to time lag n∆t. The different type of motion associated with the lateral diffusion of a protein were classified by fitting plots of MSD vs. n∆t to the models for normal diffusion, hindered diffusion, and corralled diffusion:36



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〈 (∆)〉 = 4(∆)

Normal Diffusion

(4)

〈 (∆)〉 = 4(∆)

Hindered Diffusion

(5)

Corralled Diffusion

(6)

〈 (∆)〉 = 〈 ! 〉 "1 − % '(

)* (∆) , 〈+ 〉

Where D is the diffusion coefficient, 〈 ! 〉 is the corral size, and % and % are constants determined by the shape of the corral. If α < 1, the diffusion is considered to be hindered. If the track yields a maximal mean squared displacement the protein is corralled within a small lipid phase domain. If the mean squared displacement increased linearly with time lag then the protein diffusion is consider normal (Brownian diffusion). All individually tracked proteins fell under the category of normal diffusion. There were no signs that CPR was corralled or the diffusion was hindered. A control experiment was also carried out to estimate the photochemical bleaching rate of the probed attached to CPR. In this experiment labeled CPR was nonspecifically adsorbed to the glass coverslip and labeled CPRs were tracked until they bleached. The lengths of these tracks were collected in a histogram and fit to an exponential decay curve (Figure S2; Supplementary Material). The bleaching rate of the probe was measures to be approximately kbleach = 0.56 ± 0.04 sec-1. 3. RESULTS 3.1 Characterization of a Biomimetic for the Endoplasmic Reticulum (ER) FRAP curves and fluorescence images for the ER biomimetic at 17 oC, 25 oC, and 37 oC are depicted in Figure 1. The temperature dependence of the diffusion coefficient (D) as well as


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an Arrhenius plot is given in Figure 2. The value of D and its standard deviation at each temperature point was determined by averaging the results from three different samples. The value of D at each temperature point for each individual sample was the average of 10 FRAP measurements on different parts of the lipid bilayer. From these experiments four important observations were made: (1) the fluorescence images showed no signs of heterogeneity throughout the sample, (2) the individual FRAP curves all recovered to 80% or higher, indicating very little interaction between the fluorescent probe in the bottom leaflet of the bilayer and the underlying substrate, (3) the D vs. T curve smoothly decreases with temperature with no observable phase changes in this temperature range, (4) and an Arrhenius plot (-ln(D) vs 1/T) gave a straight line with an activation energy of 52.7 ± 0.6 kJ/mol. Please note that it has been demonstrated that the temperature dependence of the of a diffusion coefficient is governed by the Arrhenius equation.37-38 Together these results strongly indicate that the cushioned planar supported ER biomimetic membrane has a single homogeneous phase from 17 oC to 41 oC.

3.2 Catalytic Activity of CYP2C9/CPR in the Biomimetic ER For the biomimetic ER to represent a functional reconstitution system for the cytochrome P450 machinery, the metabolism of diclofenac by CYP2C9 in the presence of excess CPR was evaluated. In this way, the functionality of CPR was also assessed, being the only electron donor for P450 to perform catalysis. Activity was found in both ER liposomes and planar supported lipid bilayers indicating that both P450 and CPR are functional in the biomimetic. It was not possible to perform a saturation curve for planar supported bilayer, because at low substrate concentration the amount of metabolite formed was close to the detection limit of the instrument. However, we could detect the chromatographic peak corresponding to the MRM transition of the



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metabolite when saturating concentrations of diclofenac was used (Figure S1, Supplemental Information). For ER liposomes, saturation kinetics was performed and the corresponding kcat and KM values were 2.8 ± 0.2 min-1 and 9.8 ± 0.9 µM, respectively (Figure 3). It is important to note that these experiments were performed without detergent present, setting them apart from other reconstitution systems.

3.3 Diffusion of Cytochrome P450-Reductase in the Absence of NADPH Attempts to use FRAP to measure the diffusion of CPRox within the ER biomimetic were unsuccessful. Under the conditions described in section 2.6 but with the concentration of CPRox raised to 7.5 nM, a significant portion of labeled CPRox was observed to exist in the solution phase. This was evidenced by imaging the sample at the bilayer and then far up into the solution layer. After successive rinsing steps this phenomenon persists, though at decreased concentrations of labeled CPRox. The high background signal from the solution phase prevented the measurement of a well-defined bleaching and recovery area at the membrane. This prevented the measurement of the diffusion coefficient of membrane bound CPRox by FRAP. As described below this is due to CPRox existing in equilibrium between solution phase and a membrane-associated state. In order to test this hypothesis, single molecule tracking of CPRox was carried out in an attempt to directly measure CPRox binding to the membrane from the solution phase and to determine the diffusion coefficient associated with membrane bound CPRox. Representative tracks of oxidized ligand-free CPR (CPRox) in the ER biomimetic are depicted in Figure 4a along with plots of their MSD vs. lag time and individual diffusion coefficients. It is immediately apparent that at 37 °C CPRox diffuses without any noticeable


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confinement or hopping with the protein diffusing rapidly through the field of view. A second observation is that CPRox would often appear in the field of view and begin to diffuse normally instead of always diffusing in from the edge. The single protein tracking experiments described in this study are 2-D and use an evanescent field that can only excite molecules within the membrane; proteins in solution are not observable. The result is that membrane associated protein can suddenly appear from solution within the field of view and diffuse normally until they reenter the solution phase. In contrast, integral membrane proteins can only diffuse in from the side of the field of view. In this work, it was observed that CPRox behaves more like a membrane associated protein in equilibrium with the buffer rather than a purely integral membrane protein. Diffusion coefficients were obtained for particles that could be tracked for 10 frames (250 ms) or longer. Nearly all MSD vs. time lag curves for individual proteins were linear in nature and therefore fit using the normal diffusion model (eq. 4; Figure 4a). The same was true for the average MSD vs. time lag (Figure 4b). Depicted in Figure 4c is the probability distribution of diffusion coefficients derived from individual CPRox tracks. The probability distribution is simply a normalized histogram of the individually measured diffusion coefficients. The bins of the histogram were set by the Freedman-Diaconis rule.39 In a detailed Monte Carlo study, Saxton and co-workers carefully worked out the factors responsible for the shape of a histogram of diffusion coefficients.40 They found that linear least squared fits of MSDs vs. time lags for tracks greater than 64 frames gave Gaussian distributions centered at the value for D. For shorter tracks, less than 64 frames, they found that the leading edge is steeper than a Gaussian, has a peak modestly lower than D, and a longer trailing edge. However, they also observed that the average diffusion coefficient equaled the value set by the simulation. It has also been shown that the distributions acquired from short tracks can be fit to a gamma



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distribution.36, 41-43 In the current study, all histograms were fit to a single gamma distribution. There were no discernable asymmetry or obvious plateaus. For CPRox in the absence of NADPH the measured diffusion coefficient from the average MSD vs. time lag (DaMSD = 2.2 ± 0.2 µm2 s1

) closely corresponded to the mean diffusion coefficient determined from the gamma

distribution (DΓ

= 2.5 ± 0.2 µm2 s-1).

Control experiments using solid supported membranes composed of 1:1:1 DOPC:DLPC:DLPS by weight were also carried out. In these control experiments, virtually all the CPRox was washed from the membrane after the sixth rinsing step of the procedure. The tiny fraction that did remain produced occasional transient interactions with the membrane lasting a single frame on average. These results indicate the association of CPRox with the ER membrane is much stronger than what is observed for the DOPC:DLPC:DLPS membrane that is typically used as a reconstitution system for CPR/P450s. It should also be noted that without the use of a detergent, this reconstitution system shows reduced activity.27 These results are consistent with that earlier reported observation.

3.4 Lateral Diffusion of Cytochrome P450-Reductase as Modulated by NADPH Binding NADPH binds to the FAD/NADPH domain of CPR.44-45 According to Roberts et al.,5 under anaerobic conditions stochiometric amounts of NADPH causes a 2e- reduction of CPR (CPR2-) by means of a hydride transfer, whereas an excess amount of NADPH further reduces the protein to the 4e- state (CPR4-).46 Fully reduced CPR4- is thermodynamically unstable and rapidly oxidizes in the presence of oxygen.47-48 Therefore CPR2- is normally considered the state leading to electron transfer to P450’s,46 but for completeness the fully reduced CPR was also


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investigated. To understand the effect of NADPH on the lateral mobility of CPR in the ER biomimetic, two experiments were carried out. The first was with stoichiometric amounts of NADPH (450 pM) and the second was with a 5-fold excess of NADPH to CPR (2.25 µM). Anaerobic conditions were maintained during the entire course of the experiments through the oxygen scavenging system described in section 2.6 and by encasing the microscope in a sealed chamber with a constant flow of dry N2(g). Figure 5a depicts representative tracks and corresponding individual MSD vs. time lag plots of CPR diffusing in the ER membrane when stochiometric amounts of NADPH were present (CPR2-). The presence of the ligand and subsequent reduction of the FAD/FMN domains caused: (1) a near complete partitioning of the protein into the membrane, (2) longer tracks that correspond to this partitioning, and (3) a much slower diffusion coefficient. The first observation was based on the fact that CPR2- did not appear from solution like CPRox, but diffused in from the edge of the field of view. A large majority of the tracks (>95%) were best fit with a normal diffusion model (eq. 4), and the analysis of the average MSD vs. time lag confirmed that CPR-2 diffuses normally (Figure 5b). Diffusion coefficients were determined as described in the previous section using both the average mean squared displacement (DaMSD = 1.4 ± 0.1 µm2 s-1) and the mean value of the probability distribution (DΓ

= 1.5 ± 0.1 µm2 s-1).

As was the case for

CPRox, the values of DaMSD and DΓ for CPR2- closely match one another. Finally, the value of D for CPR2- is ~40% lower than what was measured for with CPRox. Fully reduced CPR (CPR4-) was achieved by increasing the molar ratio of NADPH:CPR to 5:1. Under these conditions, protein diffusion coefficients decreased further and remained strongly partitioned in the membrane. The data are presented in Figure 6. The MSD vs lag time



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curves are linear (normal diffusion) and DaMSD vs. DΓ closely match one another (DaMSD = 1.3 ± 0.1 µm2 s-1; DΓ

= 1.3 ± 0.1 µm2 s-1). The diffusion coefficient of CRP4- is ~56% slower than

observed in CPRox. 4. DISCUSSION CPR has an N-terminal membrane-binding domain (MBD) that is believed to function as an anchor to the membrane. The MBD has an acetylated N-terminus, a hydrophobic region with an α-helix as its predicted structure,49 a stop sequence, and flexible segment that is susceptible to proteolytic cleavage. According to Black and Coon,50 there are two possible orientations of CPR in the membrane. One in which the N-terminus is on the side of the membrane that is opposite the C-terminus and the other which the N- and C-terminus are on the same side of the membrane. Described in this study are the first direct observations of individual CPR proteins as they interact with and diffuse through a planar supported lipid bilayer that closely matches the chemical composition of the endoplasmic reticulum found in humans. The experimental results add to the model for CPR-membrane interactions by describing the role of NADPH. The results point to a model in which the interaction of CPRox (ligand free) with the ER biomimetic is best described as a peripheral protein in equilibrium with the buffer above the membrane. This is based on the observations that CPRox has a relatively large diffusion (DΓ = 2.5 ± 0.2 µm2 s-1) coefficient in comparison to the reduced forms and the equilibrium between a membranepartitioned state and a solution state can be directly observed. After NADPH-mediated reduction of CPRox to CPR2- and then to CPR4-, the mass transport properties dramatically change: (1)


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equilibrium between the reduced forms of CPR in the membrane and the buffer is absent from experimental observations, (3) the length of the tracks increase as would be expected for proteins that do not return to the solution phase, and (3) the diffusion coefficients decrease by 40-56% in comparison with CPRox. These observations are consistent with the reduced forms of CPR existing as integral membrane proteins as predicted by Black and Coon. A depiction of the different protein-lipid states is depicted in Figure 7 and discussed further below.

4.1 A Functional ER Biomimetic for Studying Protein / Lipid Interactions at the SingleMolecule Level. Reconstitution of cytochrome P450 activity in artificial or biomimetic systems goes back to the very beginning of P450 research. The Coon51 and Ingelman-Sundberg52-53 research groups pioneered the investigation of the role of phospholipids for protein incorporation and activity, showing that (1) a phospholipidic environment is essential for CPR-mediated electron transfer and thus cytochrome P450 catalysis and (2) protein / lipid interactions are governed by the electrostatic nature of the phospholipids. From simple phosphatidylcholine / protein mixture,52 the reconstitution of P450 activity has evolved to more heterogeneous systems, such as the broadly used ternary system composed of DOPC:DLPC:DLPS in a ratio 1:1:1 (w/w/w) with differing amounts of detergent as described by Shaw et al.27 Although not representative of the ER composition, they worked out the optimal conditions for metabolic activity of several P450 isoforms, including CYP3A4 and CYP2C9. Minor components of the lipid fraction, such as sphingolipids and cholesterol, have been related to catalytic efficiency of cytochrome P450 system,24, 54 and are becoming more and more important in defining how CPR and P450 perform their biochemical activity in their natural environment.



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More importantly, the systems described above provide scarce insights about the organization of P450 and its redox partner CPR in the membrane, as well as the protein – protein interactions associated with the transfer of electrons between CPR and P450s. The Nanodiscs® developed by Sligar’s group are more sophisticated tools, although they confine the protein in a narrow membrane landscape that make it difficult to study processes that are associated with mass action.55 The biomimetic ER developed in this study essentially fills the gap between the demand of a system that concurrently reflects the natural environment of these proteins, is still able to guarantee metabolic activity, and is suitable for protein – lipid and protein – protein interaction studies. When developing a new constitution system there are several concerns that first need to be addresses. These include: (1) Can the protein of interest be reconstituted into the membrane? (2) Is the reconstituted protein still active? (3) Is the membrane heterogeneous and if it is heterogeneous how does it affect the attachment, partitioning and distribution of the protein within the membrane? (4) What phase (liquid, gel, mixed, etc) is the membrane in at the temperature of the biophysical experiments? (5) If it is a supported planar membrane, is there a large immobile fraction of protein stuck to the solid support? As discussed below, CPR can be reconstituted into the ER biomimetic and is active in both liposomes and planar supported bilayers. Fluorescence micrographs have shown that the ER membrane is homogeneous (Figure 1). Therefore, individual domains or domain boundaries do not govern the association of CPR to the ER membrane. The FRAP experiments show that the ER exists as a single liquid crystalline phase from 17 oC to 41 oC (Figures 1 and 2), therefore CPR dynamics are not hindered by any gel phase domains that could lead to immobility, intermittent mobility, or hindered diffusion. Since it has been demonstrated that proteins incorporated into solid supported bilayers (i.e. on


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glass or quartz supports) are often immobile and have altered or diminished protein functionality, the ER mimic was cushioned by a PEG layer35, 56 and shows no immobile fraction. An essential requirement for an artificial system is to preserve the biochemical properties of the enzyme being studied. Although this work is focused on CPR, the ongoing project in our laboratory has a wide-ranging interest in cytochrome P450. Measuring CYP2C9-induced oxidation of diclofenac allowed for the evaluation of the activity of the P450 / CPR redox couple in our biomimetic ER. When measured in SUVs, the saturation plot gave a catalytic affinity (KM) analogous to the one observed for insect cells and yeast microsomes (3-15 µM).32, 57 Locuson et al.58 reported a kcat of 8-10 min-1 for CYP2C9 diclofenac metabolism when reconstituted in DLPC liposomes; however, the P450:CPR ratio used (0.2:16) was considerably higher than the present study (1:2). In a second study Yamazaki et al.,59 carried out a series of experiments in which the source of the CYP2C9 was either from lymphoblastoids, baculosomes, supersomes, and E. coli membranes and were reconstituted into 1:1:1 (by mass) DLPC:DOPC:DLPS with 0.25 mM sodium cholate at a CYP2C9:CPR ratio of 1:4. They observed catalytic activities for CYP2C9-mediated hydroxylation of diclofenac was highly dependent on the source of the enzyme with kcat values that ranged from 2.4 – 23 min-1. Brignac-Huber et al.24 were also able to show catalytic activity of CYP1A2 in a similar ER system, using fluorescence-based activity assays. It is important to point out that for the experiments described in this article no detergent was added before incorporation, and the purified protein was dialyzed overnight in the presence of silica beads to remove any residual detergent from purification.

4.2 CPRox is a Peripheral Membrane Protein



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Early observations by Ingelman-Sundberg53 and reviewed more recently by Reed and coworkers,60 pointed out the inherent difficulties in incorporating CPR alone with preformed vesicles. Since then the standard preparations for reconstituting CPR is to either incubate the protein in vesicles that contain pre-incorporated P450s,61 or by co-incubating both proteins with empty vesicles.13, 24 In both strategies, membrane incorporation is facilitated by a small amount of detergent (cholate, CHAPS, ect.), which is subsequently removed by dialysis or incubation with silica beads.24, 60 Although CPR is believed to be anchored by its flexible N-terminal MBD to the ER membrane,5-8 the transmembrane nature of full-length CPR (as well as of human cytochrome P450s) has never been proven with structural evidence. The first X-ray crystal structure of the FMN-binding domain of human CPR was provided by Zhao et al.,62 and several others structures for both human and rat CPR were later published.5-7 In all these structures, the MBD is cleaved to allow complete solubility and subsequent crystallization; the lipids were not present in these structures. As a result, the published structures are devoid of any information pertaining to the molecular organization of CPR in the membrane. Recently, Huang et al. performed solid-state NMR experiments using the functional FMN binding domain of CPR, probing that the N-terminus MBD adopts a α-helix conformation in DLPC/DHPC/cholesterol bicelles.49 Nevertheless, the lack of the complete proteinic structure does not allow any intramolecular interaction of the flexible N-terminus domain. In contrast, the single-molecule techniques used in this study rely entirely on the direct observation of individual full-length proteins as they interact with the lipid bilayer. The results presented here complement the structural work and can partially explain the intrinsic difficulties in incorporating CPR alone in vescicles.53, 60 The ER biomimetic used in this study is able to “trap” a certain amount of CPRox, as the presence of diffusing protein indicates. The lipid composition also seems to greatly affect


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protein / lipid interactions. For example, anionic lipids have been associated with higher interaction of CPR7 and a more negative redox potential.15 The chemical composition of the ER biomimetic in this study readily promotes detergent free incorporation but the oxidation state of CPR (presence of NADPH) determines whether it is a peripheral or integral membrane protein.

4.3 Reduction of CPR by NADPH Modulates Interaction with the ER Membrane. The mechanism of NADPH binding to CPRox and subsequent electron transfer to the FAD and FMN domains has been the subject of several studies and the detailed kinetic mechanism of this process has been revised and reviewed many times.3-4 Moreover, it has been shown that there are large conformational changes between the FAD and FMN domains as this process is carried out.5-6, 63 Briefly, the conformation in which the FAD and FMN domains are furthest apart is said to be open. When the domains are in its closest conformation it is said to be closed. A simplified electron transfer mechanism is as follows: (1) membrane bound CPRox is in an open configuration and ready to accept a NADPH ligand, (2) NADPH binds to CPRox and transfers two electrons through a hydride ion transfer which produces CPR2- in a closed conformation, and (3) NADP+ is released returning CPR2- to an open conformation.5 If NADPH is in low concentration the electron transfer mechanism ends and CPR2- is ready to transfer a single electron to a number of redox partners (P450s are its major partners). If under anaerobic conditions and in the presence of excess NADPH, CPR2- in its open configuration can accept a second NADPH ligand which reduces CPR2- to CPR4- by a second hydride ion transfer. Presumably, this would be accompanied by a closed conformation. CPR4- could then return to an open conformation by releasing a second NADP+ ion.1, 3-4



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In the experiments described in this article, the samples are devoid of a redox partner and CPR is allowed to equilibrate with NADPH for a long time (>1.5 hr). Under these conditions, we can be confident that NADP+ has had enough time to be released from the protein. Therefore CPR2- (stoichiometric amount of NADPH) and CPR4- (excess NADPH) would be in their open conformations and may carry a negative charge on one or both of the prosthetic FAD and FMN molecules. (Note: the protonation states FMN and FAD when CPR is in its reduced states are unknown.) It is reasonable to assume that conformation and redox state of CPR can have a big effect on its interaction with the membrane. While no structural studies were carried out, it is clear that NADPH has a profound effect on how CPR interacts with the membrane. 4.4 Possible Impact of CPR dynamics on P450 Reduction. As mentioned in the introduction, one striking characteristic of the P450 kinetic machinery is the physiological non-stoichiometric ratio of CPR and the monooxygenase components. This has raised the question of how so few CPRs can efficiently reduce many more P450s. Previous hypotheses relied on the notion that CPR behaved exclusively as an integral membrane protein and therefore diffused two-dimensionally. This gave rise to the hypotheses that P450s could form dimers with increased affinity for CPR13 and the formation of ‘functional clusters’ in which a single CPR can serve several P450s through an electron hopping mechanism between members of the cluster.11,12 While the observation that CPRox exists as a peripheral membrane protein does not invalidate earlier hypotheses pertaining to the reduction of its redox partners, it could give rise to other hypotheses or augment existing ones. One new hypothesis that could lead to increased efficiency of P450 reduction by CPR takes into account that reaction rates are phenomenologically limited by how fast reactants can come into contact with one another. This


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study has shown that in the peripheral membrane state, CPRox can diffuse quickly when associated with the membrane and can hop from place to place as it goes back and forth between solution and membrane. We suggest that this could allow CPRox to efficiently find its redox partner and form a transient complex. Reduction of CPRox by NADPH to yield CPR2- would then generate the integral membrane state in close proximity to its redox partner. This would then give CPR2- enough time to efficiently transfer its electrons to P450 and bring CPRox back to its peripheral membrane state. Once its cycles back to CPRox it could dissociate from the membrane and find the next P450 enzyme. Studies are underway to test this hypothesis by incorporating CPR and CYP2C9 into the ER membrane and directly observing protein-protein dynamics as a function NADPH concentration. 5. Conclusions The most important conclusion from this work is that NADPH mediates the interaction of CPR with a planar supported bilayer that was designed to mimic the endoplasmic reticulum. Without NADPH, CPR (CPRox) behaves as a peripheral protein with a large diffusion coefficient (DΓ

= 2.5 ± 0.2 µm2 s-1) and in equilibrium with the solution phase.

The introduction of

NADPH transforms CPR to an integral membrane protein with much smaller diffusion coefficients. The measured diffusion coefficient for CPR4- (DΓ smaller than that measured for CPR2- (DΓ

= 1.3 ± 0.1 µm2 s-1) is a little

= 1.5 ± 0.1 µm2 s-1) indicating that the former

interacts more strongly with the ER biomimetic. These observations may help account for the versatility of CPR as an electron-transfer catalyst for cytochrome P450. It is also important to note that the membrane described here has the same lipid composition as a natural human ER with the exception of a PEG cushion, both CPR and CYP2C9 will self-insert into the membrane



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without any detergent present, and form a redox pair capable of enzymatic activity comparable to that observed in microsomes.

ASSOCIATED CONTENT Supporting Information Data for activity assay in a planar supported ER bilayer, histograms of track lengths, and bleaching time. This material is available free of charge via the Internet at: http://pubs.acs.org

Acknowledgements This work was supported by the NIH grant NIGMS GM114396 (J.A.B.)


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Figure Captions Figure 1. FRAP curves and micrographs of the ER biomimetic with 0.01 mole percent Rhodamine-DMPE at 17o C (top), 25o C (middle), and 37o C (bottom). The diameter of the bleached area was 3.3 µm and recovered to >80%. Figure 2. Temperature dependence of the diffusion coefficient for Rhodamine-DMPE in the ER biomimetic (top) and corresponding Arrhenius analysis (bottom). Figure 3. Saturation kinetics plot for oxidation of diclofenac using ER liposomes containing purified CYP2C9 and CPR (1:2 protein ratio). Each point represents an average ± standard deviation for three replicates. Figure 4. Single protein tracking experiments carried out in the absence of NADPH at 37o C. (a) Representative examples of single CPRox proteins diffusing in the ER biomimetic (top) and the corresponding MSD vs. time lag curves for individual proteins. N = the total number of frames the protein was tracked before it disappeared and D = the diffusion coefficient associated with the individual protein. (b) Average MSD vs. time lag for all measured tracks. DaMSD = the diffusion coefficient determined from the average MSD vs. time lag curve. (c) Measured probability distribution (normalized histogram) of individually measured diffusion coefficients (bars). Solid black line is a fit of the probability distribution to a gamma distribution. DΓ = mean diffusion coefficient derived from gamma distribution. Number of Tracks = total number of tracks that make up the histogram. Figure 5. Single protein tracking experiments carried out in the presence of stoichiometric amounts of NADPH at 37o C. (a) Representative examples of single CPR2- proteins diffusing in the ER biomimetic (top) and the corresponding MSD vs. time lag curves for individual proteins. N = the total number of frames the protein was tracked before it disappeared and D = the diffusion coefficient associated with the individual protein. (b) Average MSD vs. time lag for all measured tracks. DaMSD = the diffusion coefficient determined from the average MSD vs. time lag curve. (c) Measured probability distribution (normalized histogram) of individually measured diffusion coefficients (bars). Solid black line is a fit of the probability distribution to a gamma distribution. DΓ = mean diffusion coefficient derived from gamma distribution. Number of Tracks = total number of tracks that make up the histogram. Figure 6. Single protein tracking experiments carried out in the presence of excess NADPH at 37o C. (a) Representative examples of single CPR4- proteins diffusing in the ER biomimetic (top) and the corresponding MSD vs. time lag curves for individual proteins. N = the total number of frames the protein was tracked before it disappeared and D = the diffusion coefficient associated with the individual protein. (b) Average MSD vs. time lag for all measured tracks. DaMSD = the diffusion coefficient determined from the average MSD vs. time lag curve. (c) Measured probability distribution (normalized histogram) of individually measured diffusion coefficients (bars). Solid black line is a fit of the probability distribution to a gamma distribution. DΓ = mean diffusion coefficient derived from gamma distribution. Number of Tracks = total number of tracks that make up the histogram.



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Figure 7. Sketch depicting the interaction of CPR with the ER biomimetic membrane as a function of reduction by NADPH. The membrane binding domain (MBD) outlined in pink is an artist’s rendition of a generic MBD containing an α-helix and β-sheet motif that anchors the CPR to the membrane.49-50 The MBD is depicted with the C-terminus on the same side of the membrane as the FMN FAD/NADPH domains, but it is also possible that the C-terminus could be on the opposite side of the membrane. The conformational states depicted in the sketch are based on the combined work from references 2-4. In the oxidized form, CPRox is a peripheral protein in equilibrium with the solution phase. After the reduction by a hydride ion transfer and the release of NADP+, CPR2- becomes an integral membrane protein. After further reduction and release of a second NADP+ cation, CPR4- is also an integral membrane protein.


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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Figure 7



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Table of Contents Figure


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