Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii

Abstract

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λ_max = 390 nm; D380N, λ_max = 506 nm) and one with red-shifted (D380E, λ_max = 533 nm) absorption maxima relative to the wild-type protein (λ_max = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

Keywords: adenylate cyclase (adenylyl cyclase); cyclic nucleotide; optogenetics; photoreceptor; rhodopsin; spectral tuning.

Figure 1.

Predicted domain structure and transmembrane topography for RhoGC. The domains are as follows: N-term, N-terminal domain; rho, microbial type I rhodopsin domain; CC, coiled-coil domain; and GC, guanylyl cyclase domain. Transmembrane helices were predicted and drawn by Protter (wlab.ethz.ch/protter/start (25); please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party-hosted site). The amino acid residues are as follows: red, Glu-254 and Asp-380 counterions targeted for mutagenesis in the spectral tuning experiments; blue, Lys-384, presumed site of covalent attachment to the chromophore; yellow, Glu-497 and Cys-566, residues controlling the specificity for the GTP substrate.

Figure 2.

SDS-PAGE and Western blotting analysis of fractions obtained during immunoaffinity purification of RhoGC from transfected HEK293-GnT1⁻ cells. Shown are fractions from the purification of RhoGC with the C8 antibody. A, Coomassie-stained gel. B, Western blot of the same fractions as in A using the 1D4 antibody. M, molecular mass markers; L, load (post-nuclear supernatant fraction); FT, flow-through (non-bound fraction); W1, first wash; FW, final wash before elution; E1, first elution with C8 peptide; E2, second elution with the C8 peptide. FL indicates the position of FL RhoGC on the gel.

Figure 3.

Purification of full-length (FL) and truncated (T) forms of RhoGC by tandem immunoaffinity chromatography on C8- and 1D4-antibody columns. A, Coomassie-stained gel of fractions from tandem immunoaffinity column purification using the C8 antibody first followed by the 1D4 antibody column. B, Coomassie-stained gel of fractions from tandem immunoaffinity column purification using the 1D4 antibody first followed by the C8 antibody column. C, Western blot of fractions using the C8 antibody as probe. D, Western blot of fractions using the 1D4 antibody as probe. Identifiers common to all panels: M, molecular mass markers; FL, full-length RhoGC; T, truncated RhoGC missing the N-terminal domain; GC, guanylyl cyclase domain; and N-term, the N-terminal domain. Purification presented in panel A: C8, eluent from the C8 column; FT, flow though, or non-bound fraction, from the C8 eluent applied to the 1D4 column; 1D4, eluent from the 1D4 column. Purification presented in panel B: 1D4, eluent from the 1D4 column; FT, flow though, or non-bound fraction, from the 1D4 eluent applied to the C8 column; C8, eluent from the C8 column. Fractions in panels C and D are from single-step purification using the C8 and 1D4 antibody columns: lane 1, eluent 1 from C8 column; lane 2, eluent 2 from C8 column; lane 3, eluent from 1D4 column; lane 4, SDS elution from the C8 column (intense band running with a mobility of about 25 kDa is the light chain of the C8 antibody). Molecular mass marker lanes were spliced as indicated by the dividing line. Note that the gels in panels A and B are 10% polyacrylamide while those in panels C and D are 12 % polyacrylamide, which accounts for slight variation in the observed electrophoretic mobilities, particularly with respect to the N-terminal fragment.

Figure 4.

Absorption spectra for RhoGC expressed in and purified from transfected HEK293-GnT1⁻ cells. A, FL RhoGC purified by tandem immunoaffinity chromatography on C8 and 1D4 antibody columns. B, truncated RhoGC purified from the non-bound fraction of a 1D4 column after loading C8-purified RhoGC. The spectra have been normalized for absorbance at 280 nm.

Figure 5.

Light-dependent guanylyl cyclase activity of RhoGC in membranes isolated from transiently transfected HEK293-GnT1⁻ cells. Each reaction contained 0.85 μm RhoGC, as determined by Western blotting analysis of the membranes. The formation of cGMP from GTP (5 mm) was followed by HPLC. The reaction was allowed to proceed for 10 min in the dark, after which the reaction was exposed to light (first arrow) from a 300-W tungsten bulb filtered through a 475-nm cut-on filter. The reaction was returned to the dark at 20 min and exposed to light again at 30 min, as indicated by the second and third arrows, respectively.

Figure 6.

Substrate depletion assay for reaction of FL RhoGC with [α-³²P]GTP in detergent solution. A, the reaction consisted of 1 μm RhoGC and 1 mm [α-³²P]GTP (0.02 Ci/mmol) in 50 mm Tris buffer (pH 8.0) containing 50 mm NaCl, 10 mm MgCl₂, 0.5 mm EDTA, and 0.1% DM (w/v). B, the same as in A, except the reaction also contained 1 mm cGMP product. The reaction was initiated by exposure to continuous illumination from a 300-W tungsten lamp filtered through a 475-nm cut-on filter. The data (discrete points) were fit (solid line) to the integrated form of the Michaelis-Menten equation using the COPASI biochemical system simulator software (http://www.copasi.org (26, 27); please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party-hosted site), from which the K_m and k_cat for the enzyme were determined.

Figure 7.

Characterization of the radioactivity-based assay for guanylyl cyclase activity in detergent solution. A, dependence of enzyme activity on light. On and off arrows indicate the times at which the 300-W tungsten bulb was turned on and off, respectively. B, comparison of the Michaelis-Menten curve (solid line), calculated using k_cat and K_m from the substrate depletion assay (Fig. 6), with initial rate data (data points) from reactions under the same conditions but starting with different initial concentrations of the GTP substrate. C, comparison of raw PhosphorImager images of PEI-cellulose plates for assays of the FL and T forms of RhoGC.

Figure 8.

A–F, time-resolved spectral changes for FL (A–C) and truncated (D–F) forms of RhoGC. A and D, time-resolved difference spectra recorded every millisecond for the first 1.1 s of reaction following a 5-ns pulse from a 532-nm laser (25 mJ/pulse). Time is encoded by color, as indicated in the inset. Selected spectra were omitted for clarity. B and E, heat maps for time-dependent evolution of spectral changes. Relative absorbance is color-coded as illustrated in the side bar. C and F, single-wavelength time course for the reaction monitored at 380 nm. Experimental data are shown as discrete points, whereas the fit using Equation 1 (see “Experimental Procedures”) with the indicated time constants is shown as a solid line. All data were collected at 25 °C in DDM solution.

Figure 9.

Determination of RhoGC transmembrane topology and orientation. A, immobilized CHO K-1 cells were transfected with the dual (C8 and 1D4) epitope-tagged RhoGC on glass coverslips and incubated with the antibodies under permeabilized or unpermeabilized conditions for immunofluorescent staining as indicated and as described under “Experimental Procedures.” Both C8 and 1D4 antibodies react with RhoGC, but only after the cells are first permeabilized with detergent, indicating that the N and C termini are located intracellularly. B, a higher-magnification view of the 1D4-stained cells, clearly showing a diffuse staining pattern typical of proteins localized to the plasma membrane.

Figure 10.

Normalized absorption spectra for spectral tuning mutants. In order of increasing λ_max, shown are E254D (orange), D380N (cyan), WT (black), and D380E (magenta). Absorption maxima were determined for each dataset from the derivative of a second-order polynomial fit to the data over a limited wavelength range surrounding λ_max in the visible region of the spectrum: E254D (510–480 nm), λ_max = 490.1 ± 0.2; D380N (540–480 nm), λ_max = 509.3 ± 0.1; WT (550–510 nm), λ_max = 527.4 ± 0.1; and D380E (555–520 nm), λ_max = 536.3 ± 0.3. The precision with which the λ_max values were determined reflects not only noise in the original spectrum but also the wavelength range (i.e. number of data points) used for the polynomial fit.

Figure 11.

Adenylyl cyclase mutants. A, determination of substrate specificity for WT RhoGC and the E497K, C566D, and E497K/C566D mutants. Reactions contained 0.85 μm RhoGC (WT or mutant) in membranes from transfected HEK293 cells and either GTP or ATP at 5 mm initial concentration, as indicated. Time points were collected over the first 10 min in the dark and then (arrow) under continuous exposure to light from a 300-W tungsten bulb with a 475-nm cut-on filter over the next 20 min. Cyclic nucleotides (cGMP and cAMP) were followed by HPLC as described under “Experimental Procedures.” The E497K/C566D double mutant displays some constitutive activity in the dark but has clearly had its substrate specificity changed to that of an adenylyl cyclase and exhibits 4-fold stimulation of activity by light. B, UV-visible absorption spectra for E497K, C566D, and the double mutant E497K/C566D. Spectra have been normalized to optical density = 1.0 at 280 nm.