Detergents stabilize the conformation of phosphodiesterase 6

Abstract

Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and β subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDEγ (Pγ) subunits, and binding of two types of isoprenyl-binding protein δ, called PrBP/δ, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/δ and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/δ from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.

Figure 1

Preparation of trypsinized PDE6. A. The time course of trypsin digestion monitored by SDS–PAGE is shown. Purified PDE6 was mixed with trypsin at 100:1 (weight ratio) and incubated at room temperature for 10, 20, 30, 45 and 60 min. Protein without trypsin was included as a control (Lane 0). At 30 min (arrow), a weak band with a smaller molecular weight appeared. B. Size exclusion chromatography profile of PDE6_t (30 min, room temperature). PDE6_t (solid line) eluted at nearly the same position as untreated PDE6 (dashed line). C. PDE6_t activity with cGMP (2 mM) as substrate. Enzyme activities monitored with the pH–sensitive fluorescent dye SNARF–1were positively correlated with increasing fluorescence intensity over time. PDE6_t was generated as follows: Purified PDE6 was first mixed with trypsin at a 100:1 weight ratio for 10 min. Soybean trypsin inhibitor was then added at a 1:10 mass ratio to stop the reaction (dashed line, –○–). Alternatively, PDE6 was mixed with trypsin–agarose at 30 μg protein per 1 μl agarose for 30 min and the reaction was stopped by passing the sample through a filter to remove the trypsin–agarose. Trypsinized peptides then were removed either by gel filtration (dashed line, –◇–) or buffer exchange with a 50 kDa MWCO filter (Millipore) (solid line, –•–). Activities for PDE6_t prepared by all three methods were identical.

Figure 2

PDE6 activity measurements with the pH-sensitive fluorescent dye SNARF-1. A. Diagram presenting PDE6-catalyzed hydrolysis of cyclic GMP to GMP with release of a proton. B. PDE6 activity measurements with the pH-sensitive fluorescent dye SNARF-1 in 0.1 M HEPES over a pH range from 6.8 to 8.2. The fluorescence intensity of SNARF-1 decreased with increasing pH (–•–) whereas the buffer used showed no fluorescence (–○–). C. Enzyme activity measurements of PDE6 and PDE6_t with SNARF-1. PDE6_t achieved maximal cGMP hydrolysis in about 5 min (–•–), whereas PDE6 exhibited much lower residual activity that resulted in total cGMP hydrolysis in about 60 min (-○-). The percentage of cGMP hydrolysis is plotted as a function of time.

Figure 3

Effects of additives and detergents on PDE6_t and PDE6 activity. A. Effects of reagents on PDE6_t and PDE6 activity. Activities of purified PDE6_t (upper panel) and PDE6 (lower panel) were measured in the absence (solid lines) or in the presence (dashed lines) of designated reagents. Urea (1 M, –▲–), Arginine (3 mM, –◆–), CaCl₂ (0.1M, –X–) and EDTA (5 mM, –◇–). The percentage of maximum PDE6_t activity vs. time is shown. B. Effects of selected detergents on PDE6 activity. The activities of PDE6_t (upper panel) were measured in the absence (solid line) or in the presence of specified detergents (dashed lines). C₈E₄ (10 mM, –○–), Anapoe X–100 (0.8 mM, –◇–), CHAPS (25 mM, –Δ–), n–octyl–β–D–glucopyranoside (20 mM, –◆–), n–dodecyl–N,N–dimethylamine–N-oxide (1 mM, – –) and dimethyldecylphosphine oxide (5 mM, –×–). Those detergents that did not alter PDE6_t activity were then tested for their ability to suppress PDE6 activity (lower panel). PDE6 without detergent (solid line), with detergents (dashed lines). N–undecyl–β–D–maltopyranoside (1 mM, –Δ–), C₈E₄ (10 mM, –○–), Anapoe X–100 (0.8 mM, –◇–). The percentage of cGMP hydrolyzed is plotted vs. time.

Figure 4

Effect of C₈E₄ on PDE6 activity. A. Measurements of PDE6_t (bold black line, –•–) and PDE6 (dashed line, –○–) activity in the presence of C₈E₄. PDE6 activity without C₈E₄ is shown as a solid grey line (–○–). In the presence of C₈E₄, PDE6_t was fully functional, whereas PDE6 activity was completely abolished. B. In the presence of C₈E₄, the fluorescence intensity of SNARF–1decreased with increasing pH, indicating that the dye responded appropriately to changes in pH. C. PDE6_t activity in the presence of C₈E₄. Protein was pre–incubated in the assay buffer with C₈E₄ for 10 min before PDE6_t activity was monitored (solid line). Alternatively, PDE6 was digested with trypsin in the presence of C₈E₄ before soybean trypsin inhibitor was added to stop the protease reaction and the resulting PDE6_t was added to assay buffer along with C₈E₄ for activity measurements (dashed line). Both preparations of PDE6_t were fully active.

Figure 5

SEC analysis of detergent effects on reducing PG aggregation. A. Elution profiles of three forms of PDE6 in the absence of detergent. PDE6 (short dashed line) and PDE6/δ (solid line) eluted as single peaks, whereas PG (large dashed line) eluted as two major peaks indicating a mixture of a high molecular mass aggregate and a monomer. Insert: SDS–PAGE analysis of SEC fractions from the two PG peaks. The top band is PDE6 and lower band is GST–δ. Some detergents, such as Anapoe–35 (B), reduced aggregation of PG. Insert: SDS–PAGE analysis of SEC fractions (indicated with dotted line). In the presence of C₈E₄ (C), PG aggregation was almost completely abolished. PDE6 was found mainly in Peak 1 (insert, labeled as 1). The second major peak eluting after the PDE6 peak represented GST– PrBP/δ as indicated by SDS–PAGE analysis (insert, labeled as 2). D. SEC analysis of the PDE6/δ conformation in the presence of C₈E₄; 0.5 ml fractions were collected SDS–PAGE of fractions from the two major peaks is shown in the insert. The left band (labeled with 1) shows the fraction from the main peak with an estimated MW of 200 kDa whereas the right band illustrates the fraction from the last peak with an estimated MW of 15 kDa (as indicated with an arrow). For all inserts, analysis of the protein sample before SEC is shown next to the protein marker. The top band corresponded to PDE6 and lower band to GST–δ.

Figure 6

SDS–PAGE analysis of detergent–depleted GST–PrBP/δ dissociated from the PG complex. A. C₈E₄ removed GST–PrBP/δ from the PG complex. PG was mixed with GST–FF resin and unbound proteins were removed by washing the resin with PBS. Then the resin was incubated with PBS plus 10 mM C₈E₄ for 1 hr at room temperature. The flow through was collected (Lane F, under C₈E₄) and the resin was washed again with PBS plus C₈E₄. Elution was accomplished with reduced glutathione (10 mM). F: flow through; W: wash fractions; E: Elution fraction. B. Dissociation of the PG complex was decreased by incubation with C_nE_m detergents with longer alkyl chains. C_nE_m: C_n = CH₃(CH₂)_n–1 alkyl chain, E_m = (OCH₂CH₂)mOH oligoethyleneglycol. In the presence of C₈E₄ or C8E6, PDE6 was identified mainly in SEC flow through fractions as shown by SDS–PAGE. But in the presence of C₁₂E₈, the PG complex remained intact in the elution fraction. Lanes F and E: flow through and elution fractions in the presence of C_nE_m.

Figure 7

Catalytic properties of purified PDE6_t and PDE6 assessed directly by product formation. A. Percentages of cGMP hydrolysis by PDE6_t and PDE6 are plotted as a function of time. PDE6_t: solid line. PDE6: dashed lines (2 mM, –○– and 5 nM–◇–). B. The rate of cGMP hydrolysis by PDE6_t is plotted as a function of cGMP concentration. The K_m value is 31 ± 4 μM and V_max is 5122 ± 285 mol cGMP/mol·sec. C: PDE6_t activity in the presence and absence of C₈E₄. The percentage of cGMP hydrolysis by PDE6_t is plotted as a function of time. PDE6_t without C₈E₄: solid line. PDE6_t with C₈E₄: dashed line. D. cGMP hydrolysis in the presence and absence of C₈E₄. The percentage of cGMP hydrolysis by PDE6 is plotted as a function of time. PDE6 without C₈E₄: solid line. PDE6 with C₈E₄: dashed line.

Figure 8

EM images of negatively stained PDE6/δ complexes in the absence and presence of indicated detergents. The protein retained homogeneity in all detergents tested except CHAPS in which the complex seems to have dissociated into particles of smaller size. Inset, higher magnification of a single particle. Scale bars, 100 nm.