Cloning and Expression of Proteins
AANAT 1–197 (ΔC8) and GST-AANAT 34–197 (ΔN33, ΔC8) were subcloned between the NdeI and SmaI sites of the pTYB2 intein expression vector (New England Biolabs). Then, the SmaI site was mutated (Quikchange, Stratagene) from PG to HA, which are the wild-type residues (amino acids 198 and 199) present in AANAT. Using the same methodology, a Factor Xa protease site followed by a Cys residue (IEGR-C) was introduced between the GST fusion and truncated AANAT. The resulting AANAT constructs were suitable for either two- or three-piece semisynthesis of full-length AANAT (1–207) as described in detail below. For FRET labeling, an AANAT 1Cys-199-intein-chitin binding domain was subcloned into the pET-SUMO expression vector (Invitrogen), to be used in generating a protein that could be assembled in three pieces via semisynthetic methods. In addition, full-length 14-3-3ζ was subcloned between the NdeI and SmaI sites of the pTYB2 intein expression vector. In this case, the SmaI site was left intact, resulting in the production of a full-length 14-3-3ζ protein bearing an additional PG at its C-terminus (this additional dipeptide had no effect on protein behavior).
All proteins were overexpressed in Escherichia coli BL21-CodonPlus-DE3-RIPL cells (Stratagene). Cells were grown to an OD600 of 0.6 in LB medium at 37 °C, induced with 0.2 mM IPTG, and grown for 20 h at 16 °C. Cells were harvested by centrifugation at 5000g for 15 min and resuspended in ice-cold lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM MgSO4, 5% glycerol, and 5% ethylene glycol]. The cells were then lysed via a double pass on a French press (16000–18000 psi) and clarified by centrifugation at 25000g for 30 min (4 °C). The clarified lysate was then double loaded (0.5 mL/min) onto a pre-equilibrated chitin column (New England Biolabs). The column was then washed with 20 volumes of chitin column buffer [50 mM HEPES (pH 7.5), 250 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100], followed by 10 volumes of ligation buffer [50 mM HEPES (pH 7.5), 250 mM NaCl, and 1 mM EDTA] at a flow rate of 1 mL/min. At this point, the immobilized proteins could be either cleaved from the chitin resin with DTT or ligated to a 1Cys peptide. Cleavage was afforded by overnight treatment with 1 volume of 50 mM DTT in ligation buffer at room temperature followed by extensive dialysis into storage buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 10 mM DTT]. In total, two proteins were cleaved from the resin using DTT and used for this study [ΔC8 and 14-3-3ζ ()]. Ligation will be discussed in detail below. The mass and purity of all proteins (±50 Da) were confirmed via MALDI-MS and SDS-PAGE.
Proteins prepared for this study.
All Fmoc-protected amino acids and resins were obtained from Novabiochem. All peptides were assembled using a standard Fmoc solid phase peptide synthesis strategy on a Ranin PS-3 automated peptide synthesizer. 1Cys peptides for C-terminal EPL were assembled on the Wang resin, where cleavage from the resin and global deprotection could be afforded by treatment with reagent K (87.5% TFA, 5% water, 5% thioanisole, and 2.5% ethanethiol). Two AANAT C-terminal 1Cys peptides were synthesized for C-terminal expressed protein ligation: H2N-CLRRNSDR-COOH and H2N-CLRRNpSDR-COOH. Ligation of these peptides to the C-terminus of AANAT resulted in a single conservative point mutation (A200C).
The AANAT N-terminal pT31 peptide containing an α
LPG-COS-benzyl) was synthesized by a modified Fmoc strategy with the C-terminal carboxylate derivatized in the solution phase followed by side chain deprotection with reagent K (13
). A Gly residue was added to the C-terminus of the peptide sequence to eliminate epimerization at the C-terminal amino acid and to aid with ligation. Briefly, the peptides were assembled on the weak acid labile 2-chlorotrityl resin (2-Cl-Trt); then, the fully protected α
carboxylate was cleaved from the resin with 25% hexafluoro-2-propanol, activated with diisopropylcarbodiimide, and coupled to benzyl mercaptan to yield the benzyl α
All deprotected peptides were precipitated and washed with cold diethyl ether and then lyophilized to yield crude peptide solids that were purified by preparative scale RP-HPLC using a water/acetonitrile gradient in the presence of 0.05% TFA. All peptides were obtained at >95% purity as judged by analytical RP-HPLC and MALDI-MS (data not shown).
Synthesis of Fluorescent Peptides
For C-terminal fluorescent labeling of both AANAT and 14-3-3ζ, the dipeptide H2
was synthesized (15
). Briefly, the dipeptide was assembled on the rink amide resin using an orthogonally protected Lys derivative (ε
-Dde) and Boc-Cys(Trt)-OH. After peptide assembly, the ε
-amine was selectively deprotected with 2% hydrazine and reacted with NHS-rhodamine (Pierce) in DMF containing 0.4 M N
-methylmorpholine (NMM). The peptide was cleaved from the resin, deprotected, and purified as described above.
To produce the reagent for N-terminal fluorescent labeling of AANAT, we assembled the dipeptide Fmoc-Lys(ε
-Boc)-Gly on 4-sulfamylbutyryl-AM resin (loading the resin with Fmoc-Gly was achieved by following Novabiochem’s outlined procedure). Briefly, the resin was preswollen in 10 volumes of dry methylene chloride for 1 h. During this period, Fmoc-Gly-OH (4 equiv) along with 1-methylimida-zole (4 equiv) was dissolved in a 4:1 mixture of dry methylene chloride (DCM) and dimethylformamide (DMF) under a blanket of inert gas. To this clear colorless solution was added N
′-diisopropylcarbodiimide (DIC). This solution was mixed for 5 min at room temperature, the resin drained, and the acylation mixture added to it. The resin solution was agitated for 18 h at room temperature and then drained. The resin was washed with portions of DCM, DMF, methanol, and tert
-butyl-methyl ether prior to drying for subsequent use (16
). The dipeptide was formed via standard manual peptide synthesis (deprotection with 20% piperidine in DMF, activation with HATU and Hünig’s Base).
Following peptide assembly, the N-terminal Fmoc was removed with 20% piperidine in DMF, and the free amine reacted with NHS-fluorescein (Pierce) in DMF containing 0.4 M NMM. Next, the resin-bound sulfonamide was acylated with iodoacetonitrile and cleaved from the activated resin with mercaptophenyl acetic acid. Briefly, the resin (0.1 mmol) was preswollen in dry methylene chloride. During this period, a solution was made of iodoacetonitrile (25 equiv) and diisopropylethyl amine (10 equiv) in 3 mL of DMF and 1 mL of hexamethylphosphoric triamide. This dark solution was passed thought a column of activated basic alumina to provide a clear colorless solution. The swelling solvent was drained from the resin and the activation solution added. The solution was agitated in the dark for 18 h. At the end of this period, the solution was drained, and the resin was washed with portions of DMF, tetrahydrofuran, and DCM prior to immediate cleavage.
Finally, the fluorescein-labeled dipeptide was cleaved from the activated resin with mercaptophenylacetic acid and diisopropylethylamine in dichloromethane for 18 h at room temperature. In brief, the resin (0.1 mmol) was preswollen in dry DCM; during this period, a dry round-bottom flask was loaded with mercaptophenylacetic acid (2.0 mmol) and flushed with argon. The solid was suspended in DCM (10 mL) and cooled to 0 °C. To this was added dropwise Hünig’s Base (1.0 mmol) over 5 min. This causes all of the solids to dissolve and the solution to become slightly yellow and clear. The resin was drained from the swelling solution and treated with the thiol solution. The resin bed was agitated in the dark for 18 h at room temperature. The bed was drained and the filtrate reserved. The bed was washed with three additional portions of DCM, these being pooled with the original filtrate. The organics were concentrated in vacuo to provide a crude glass. The residue was treated with 10 mL of a 95:2.5:2.5 TFA/water/triisopropylsilane (TIS) mixture to remove the terminal Lys-ε-Boc group, and the peptide was filtered through Celite to remove excess insoluble thiol and concentrated in vacuo, followed by precipitation and washing with cold diethyl ether. The crude solid was dissolved in 25% acetonitrile with 0.1% TFA and lyophilized to dryness, and the crude peptide (fluorescein-Lys-Gly-COS-phenylacetic acid) was sufficiently pure to be used directly for ligation since only αthioester product could react with the 1Cys protein. By leaving the Lys-ε-amine unmodified and preparing the MPAA αthioester, we were able to enhance solubility, since this compound would be zwitterionic under the neutral conditions required for native chemical ligation.
For anisotropy-based 14-3-3ζ competition binding studies, we selected a peptide sequence based on AANAT residues 19–38 which encompassed the pT31 14-3-3ζ binding motif (fluorescein-HN-GIPGSPGRQRRHpT
) and employed a standard Fmoc solid phase peptide synthesis strategy. Following peptide assembly, the N-terminal Fmoc group was removed with 20% piperidine in DMF and the free amine reacted with NHS-fluorescein in DMF containing 0.4 M NMM. The peptide was cleaved and purified as described above. The final purified peptide ligand was dissolved in water, and its concentration was determined by quantitative amino acid analysis (Harvard University Mi-crochemistry).
In total, seven semisynthetic proteins were prepared for this study, four by C-terminally expressed protein ligation (EPL) [ss-WT, ss-pS205, ss-GC-Insert, and ss-14-3-3ζ-Rh ()] and three using a combination of C-terminal and N-terminal ligation [ss-pT31, ss-pT31-pS205, and Fl-AANAT-Rh ()]. For C-terminal EPL (i.e., two-piece assembly), ligation was carried out with chitin resin-bound intein fusion protein for 3.5 days at room temperature in 1 column volume of 200 mM 2-mercaptoethanesulfonic acid sodium salt (MESNA) containing 2 mM 1Cys peptide (). Released (ligated) proteins were then further purified by gel filtration (Sephadex G-50 in storage buffer) to remove excess free peptide. The ligated proteins were then concentrated, and the protein concentration was determined via a Bradford assay using BSA as the standard. For C-terminal rhodamine-labeled proteins, the concentration was determined by absorbance spectroscopy (ε558 = 60000 M−1 cm−1) and confirmed by SDS-PAGE using the unlabeled protein as the standard.
For sequential three-piece assembly of semisynthetic AANAT, the GST-(Factor Xa)-AANAT 34–199 (ΔN33, ΔC8) construct was used. The first ligation, C-terminal EPL, was carried out as described above. Next, the GST tag was cleaved with 10 units of Factor Xa (GE Healthcare) per milligram of fusion protein. The reaction was allowed to proceed to ~50% completion at 4 °C in Factor Xa cleavage buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM CaCl2
, and 1 mM coenzyme A], conditions which reduced the level of formation of undesired degradation products. Factor Xa cleavage exposed an N-terminal Cys that would be used in the next ligation step. Free GST and Factor Xa were removed in a single step using a mixture of glutathione agarose and benzamidine agarose (Sigma). After purification, the proteins were extensively dialyzed into N-terminal ligation buffer [100 mM HEPES (pH 7.5), 500 mM NaCl, 5 mM EDTA, 1 mM benzamidine, and 5 mM MESNA] and then concentrated. N-Terminal ligation was achieved by mixing 12.5 equiv of peptide α
thioester with 1
Cys-AANAT in N-terminal ligation buffer supplemented with 200 mM MESNA. The reaction was allowed to proceed for 20 h (>90% completion) and then the mixture immediately purified by gel filtration (Sephadex G-50 in storage buffer with 1 mM benzamidine) which led to the removal of free peptide and also separated the unligated AANAT. Semisynthetic AANAT prepared via three-piece assembly contained an extra Gly-Cys motif inserted into the protein framework (Gly from the peptide α
thioester and Cys from the recombinant portion of the protein). From previous studies on semisynthetic AANAT, we knew that this insertion preserved the 14-3-3ζ binding elements (13
). However, to thoroughly investigate the impact of this insertion and the N-terminal semisynthetic process, we generated a recombinant construct via Quikchange mutagenesis bearing the same Gly-Cys insert.
Assembly of FRET active AANAT (Fl-AANAT-Rh) was also performed using a sequential, three-piece strategy (). In this case, we used an N-terminal Sumo fusion protein which appeared to provide improved proteolytic selectivity. Again, C-terminal EPL was carried out first to ligate H2N-Cys-Lys(ε-rhodamine)-NH2 following the procedure for EPL described above. The ligated protein was extensively dialyzed against storage buffer and then concentrated. The Sumo fusion protein was cleaved using 15 units of Sumo protease (Invitrogen) per milligram of fusion protein. The reaction was allowed to proceed to completion at 4 °C in storage buffer supplemented with 500 mM NaCl and 2 mM coenzyme A. Immediately after cleavage, the digested protein was purified by gel filtration (Sephadex G-50 in N-terminal ligation buffer). AANAT-containing fractions were pooled, concentrated, mixed with 20 equiv of the fluorescein-Lys-Gly-MPAA αthioester reagent, and allowed to react for 20 h at room temperature in N-terminal ligation buffer supplemented with 200 mM MESNA. Immediately following ligation, Fl-AANAT-Rh was purified by gel filtration (Sephadex G-50 in storage buffer) to remove the free dipeptide. AANAT-containing fractions were pooled, concentrated, and purified by coenzyme A agarose (Sigma) to remove the Sumo fusion protein. The coenzyme A agarose column was loaded and washed in storage buffer diluted 1:10 with water [5 mM HEPES (pH 7.5), 15 mM NaCl, 0.1 mM EDTA, 0.5% glycerol, and 1 mM DTT]. Pure Fl-AANAT-Rh was eluted from the column in storage buffer supplemented with 500 mM NaCl and 1 mM coenzyme A.
Semisynthesis of FRET-Labeled AANAT
Phosphorylation of Semisynthetic AANATs with PKA
To evaluate potential perturbation induced by the semisynthetic methodologies, we treated semisynthetic AANATs [ss-WT, ss-pS205, ss-pT31-pS205, ΔC8, and ss-GC-Insert ()] with protein kinase A (Promega) and included them in our in vitro characterization of phosphorylated AANATs. The proteins were phosphorylated (in storage buffer supplemented with 20 mM Mg2+ acetate and 0.2–0.4 mM ATP) overnight at 4 °C using 500 units of protein kinase A per milligram of protein. Immediately following phosphorylation, the proteins were purified by gel filtration (Sephadex G-50 in storage buffer) to remove protein kinase A and ATP. Phosphorylation was confirmed by MALDI-MS, where the incorporation of one or two phosphates resulted in a mass change of +80 or +160 Da, respectively.
Proteins chemoenzymatically phosphorylated for this study
Kinetic Analysis of Semisynthetic AANATs
To determine the effects of AANAT phoshphorylation and 14-3-3ζ interaction on acetyltransferase activity, the previously developed DTNB assay (17
) was used. Briefly, the reactions (200 μ
L) were carried out in 100 mM NH4+
acetate (pH 6.8) with 50 mM NaCl and 0.5 mM acetyl-coenzyme A (AcCoA, saturating), and the tryptamine concentration was varied (from 10 μ
M to 2.5 mM). The reactions were initiated with a final AANAT concentration of 1 nM, allowed to proceed for 2 min at 30 °C, and then quenched with 200 μ
L of 6 M guanidine HCl in assay buffer. DTNB was then added to the reaction mixture to produce a final concentration of 0.2 mM, and the absorbance at 412 nm was recorded. Reactions were linear over the time period measured and did not exceed 10% completion. End point absorbance values were converted to reaction rates using the extinction coefficient of the thiophenolate (ε412
= 13600 M−1
), and the data were fit to the following equation to yield Vmax
Vmax was converted to kcat using
The assays were repeated in the presence of 5 μM 14-3-3ζ to determine the kinetic parameters of the AANAT-14-3-3ζ complex.
Binding of Semisynthetic AANATs to 14-3-3ζ
Two anisotropy-based binding assays were developed to characterize the interaction of AANAT with the adaptor protein 14-3-3ζ. The first assay was a competitive binding assay (18
) in which AANAT displaced a fluorescently labeled AANAT-derived phosphopeptide ligand (fluorescein-HN-GIPG-SPGRQRRHpT
LPANEFR-COOH) from 14-3-3ζ. These 150 μ
L assays contained 100 nM fluorescent peptide and were conducted in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 5% glycerol. To determine the KD
of the fluorescent peptide for 14-3-3ζ, the concentration of 14-3-3ζ was varied from 0.5 to 25 μ
M. The protein and peptide were allowed to equilibrate for 30 min at room temperature before the anisotropy was recorded on a SPEX Fluoromax-2 spectrofluorometer (Instruments SA, Edison, NJ). The excitation wavelength was 492 nm (2 nm slit width), the emission wavelength 520 nm (4 nm slit width), and the integration time 0.5 s. Points represent an average of five measurements. The data were corrected by subtracting the anisotropy value of the free fluorescent peptide andnormalized to an Amax
of 1. The data were fit to the following equation to yield KD
In the displacement assays, the concentrations of fluorescent peptide and 14-3-3ζ were held constant at 100 nM and 5 μM, respectively (Kd = 3.2 ± 0.3 μM), and AANAT concentrations were varied. The proteins and fluorescent peptide were allowed to equilibrate for 30 min at room temperature before the anisotropy was recorded as described above.
We developed a more sensitive anisotropy-based binding assay utilizing fluorescently labeled semisynthetic 14-3-3ζ (ss-14-3-3ζ-Rh). This permitted the direct assessment of formation of the AANAT-14-3-3ζ complex by monitoring increases in anisotropy. These 150 μ
L assays contained 10 nM ss-14-3-3ζ-Rh and were conducted in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 5% glycerol. The AANAT concentration was varied from 0.05 to 10 μ
M. The proteins were mixed and allowed to equilibrate for 30 min at room temperature before the anisotropy was recorded on the SPEX Fluoromax-2 spectrofluorometer. The excitation wavelength was 550 nm (5 nm slit width), the emission wavelength 580 nm (5 nm slit width), and the integration time 1 s. Points represent an average of five measurements. The data were corrected by subtracting the anisotropy value of free ss-14-3-3ζ-Rh and fit to eq 3
to yield KD
In Vitro Characterization of FRET-Labeled AANAT (Fl-AANAT-Rh)
Purified Fl-AANAT-Rh was subjected to absorbance spectroscopy to determine the stoichiometric incorporation of each fluorophore. The stock protein was diluted 1:10 in storage buffer, and the absorbance spectrum was recorded from 300 to 700 nm versus a buffer blank. The molar concentration of each fluorophore was estimated from its respective extinction coefficient (rhodamine ε558 = 60000 M−1 cm−1, and fluorescein ε501 = 68000 M−1 cm−1).
To determine the intramolecular FRET efficiency, Fl-AANAT-Rh was subjected to thrombin proteolysis followed by fluorescence spectroscopy to determine the donor (fluorescein) to acceptor (rhodamine) fluorescence ratio over varying degrees of AANAT digestion. AANAT contains a single major thrombin cleavage site (3
), making this a desirable protease for the analysis. These reactions (20 μ
L) were carried out in storage buffer and contained 4 μ
g of Fl-AANAT-Rh and 0.1–10 units of thrombin (GE Healthcare). The digestions were run in duplicate and were allowed to proceed at room temperature overnight. For each thrombin concentration, one reaction was quenched with 5 μ
L of 5 × SDS-PAGE loading buffer and used to determine the extent of digestion via SDS-PAGE. The duplicate sample was diluted 7-fold with storage buffer and used for fluorescence spectroscopy on a SPEX Fluoromax-2 spectrofluorometer to determine the FRET ratio. Emission spectra were recorded from 500 to 700 nm with an excitation wavelength of 495 nm. The slit width was 1 nm for incident light and 1 nm for emission light.
CHO-K1 cells (ATCC) were cultured in ATCC F-12K medium supplemented with 10% (v/v) fetal bovine serum and 100 units each of penicillin and streptomycin and incubated at 37 °C in the presence of 5% CO2. Cells were seeded onto glass-bottom culture dishes (MatTek Corp.) and used for microinjection within 24 h (60–70% confluence). Cells were washed twice with Hank’s balanced salt solution buffer and treated with 50 μM Forskolin (Calbiochem) or 1 μM MG132 (Sigma) as indicated prior to microinjection.
For microinjection and imaging, the culture dish was warmed at 37 °C. The sample (0.35 mg/mL Fl-AANAT-Rh and 1.4 mg/mL 14-3-3ζ) was loaded into Femtotip II capillaries (Eppendorf) and injected into the cells using Eppendorf Micromanipulator 5171 and Eppendorf Transjector 5246 instruments. A minimum of eight cells were treated and imaged for each dish, and at least three dishes were imaged for each of the three conditions (untreated, Forskolin-treated, and MG132-treated).
Cells were imaged on a Zeiss Axiovert 200M microscope with a 40×/1.3NA oil-immersion objective lens and cooled charge-coupled device camera (MicroMax BFT512, Roper Scientific) controlled by Metafluor version 6.2 (Molecular Devices). Dual emission ratio imaging used a 480 DF30 excitation filter, a 505 DRLP dichroic mirror, and two emission filters (535 DF45 for fluorescein and 653 DF95 for rhodamine) alternated by a Lamda 10-2 filter changer (Sutter Instruments). The exposure time was 200 ms, and images were taken every 60 s. Fluorescent images were background-corrected by subtracting fluorescence intensities of noninjected cells or background with no cells from the emission intensities of fluorescent cells microinjected with sample. The ratios of rhodamine to fluorescein emissions were then calculated at different time points and normalized by dividing all ratios by the emission ratio at the start of the imaging experiment.
FRET efficiency was determined by acceptor photobleaching. Rhodamine was photobleached at the end of the experiment by intense illumination with a 568/55 filter for 20 min. Fluorescein fluorescence intensities before (Fda
) and after (Fd
) rhodamine photobleaching and eq 4
were used to calculate FRET efficiency: