Expressed Protein Selenoester Ligation

Abstract Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one‐pot semi‐synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi‐synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane‐associated GTPase YPT6, and site‐specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.


Materials 3
General peptide synthesis procedures 4 General procedure for acyl hydrazide to selenoester conversion 6 Ubiquitin acyl hydrazide to selenoester conversion 6 General procedure for one-pot selenoesterification, ligation and deselenization (EPSL) Synthesis of Ubiquitin-TMEM43 conjugate

Synthesis of Ubiquitin-ZAP70 conjugate
Semi-synthesis of lipidated analogues of YPT6 Folding of semi-synthetic lipidated analogues of YPT6 Semi-synthesis of Hsp27 phosphoforms Folding and CD spectra of Hsp27 phosphoforms Chaperone assay for Hsp27 phosphoforms 1 H and 13 C NMR spectra (building blocks S1 and S2)

Materials
Peptide grade dimethylformamide (DMF) was obtained from Labscan.Amino acids, coupling reagents and resins for Fmoc-solid-phase peptide synthesis (SPPS) were obtained from either Novabiochem, GL Biochem or Sigma Aldrich.Manual SPPS was performed in polypropylene syringes equipped with Teflon filters, purchased from Torviq.Analytical reversed-phase ultraperformance liquid chromatography (RP-UPLC) was performed on either a Waters Acquity UPLC system equipped with PDA eλ detector (λ = 210 -400 nm), Sample Manager FAN and Quaternary Solvent Manager (H-class) modules or a Waters System 2695 separations module with a 2996 photodiode array detector.Peptides were analyzed using an XBridge BEH 5 µm, 2.1 x 150 mm wide-pore column (C18) at a flow rate of 0.7 mL min -1 or 1 mL min -1 on the HPLC system or Waters Acquity UPLC BEH 1.7 µm 2.1 x 50 mm column (C18) at a flow rate of 0.6 mL min -1 on the UPLC system.Gradients for the UPLC system were run over five minutes with an initial 1 min equilibration step (i.e.gradient from 1-6 min) while the gradients for the HPLC system were run for 30 min with an initial 5 min equilibration step (i.e.gradient from 5-35 min).Both instruments were run using a mobile phase composed of 0.1% trifluoroacetic acid (TFA) in H2O (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B) in a linear gradient as indicated.Analysis of the chromatograms was conducted using Empower 3 Pro software (2010) and retention times (Rt min) of pure peptides and proteins are reported with the gradients specified.
Preparative and semi-preparative reversed-phase HPLC was performed using a Waters 600E Multisolvent Delivery System with a Rheodyne 7725i injection valve (5 mL loading loop) and Waters 500 pump with a Waters 490E programmable wavelength detector operating at 214, 230, 254 or 280 nm.Preparative reversed-phase HPLC was performed using a Waters Sunfire C18 column (5 μm, 19 × 250 mm) at a flow rate of 14 mL min −1 .Semi-preparative reversed-phase HPLC was performed using either a Waters XBridge-BEH300 wide-pore C18 column (5 μm, 10  × 250 mm) or Symmetry C4 column (300 Å, 5 μm, 10 mm × 250 mm) at a flow rate of 4 mL min −1 or (300 Å, 5 μm, 2.1 mm × 150 mm) at a flow rate of 0.7 mL min −1 .Ubiquitin conjugate 5 was purified on a Phenomenex Luna C18 column (100 Å, 5 μm, 10 × 250 mm) heated to 50 °C using a Waters column heater module at a flow rate of 4 mL min −1 , with the gradients as described.A mobile phase of 0.1% trifluoroacetic acid in water (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B) was used in all other cases, using the linear gradients specified.After lyophilization, peptides were isolated as trifluoroacetate salts.
LC-MS was performed either on a Shimadzu LC-MS 2020 instrument consisting of a LC-M20A pump and a SPD-20A UV/Vis detector coupled to a Shimadzu 2020 mass spectrometer (ESI) operating in positive mode, or a Shimadzu UPLC-MS equipped with the same modules as the LC-MS system except for a SPD-M30A diode array detector.Separations were performed on the LC-MS system either on a Waters Sunfire 5 µm, 2.1 x 150 mm column (C18), or wide-pore equivalent operating at a flow rate of 0.7 mL min -1 or 1 mL min -1 .Separations on the UPLC-MS system were performed using a Waters Acquity UPLC BEH 300Å 1.7 µm 2.1 x 50 mm column (C18) at a flow rate of 0.6 mL min -1 .Separations were performed using a mobile phase of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) and a linear gradient of 0-50% B over 30 min on the LC-MS System and 0-60% B over 8 min on the UPLC-MS system.
Low-resolution mass spectra were recorded on a Shimadzu 2020 mass spectrometer (ESI) operating in positive or negative mode as indicated.The deconvoluted mass spectra were generated using MagTran software. [1]Low Resolution MALDI-TOF mass spectra were recorded on a Bruker Autoflex™ Speed MALDI-TOF instrument operating in linear mode using a sinapinic acid matrix (10 mg/mL in water/acetonitrile (7:3 v/v) containing 0.1 vol.% TFA).

Loading of Hydrazide-Functionalized Resin
2-Chlorotrityl chloride resin (0.5 g, 1.22 mmol/g loading) was swollen in dry CH2Cl2 for 30 min, followed by washing with CH2Cl2 (10 x 3 mL).A solution of 5 vol.% N2H4 .H2O in DMF (5 mL) was added to the resin and allowed to shake for 30 min at room temperature.The resin was then washed with DMF (5 x 3 mL), CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL), followed by a repeat treatment with 5 vol.% N2H4 .H2O in DMF (5 mL) and washing.A capping solution of CH2Cl2:MeOH:DIPEA (17:2:1, v/v/v, 5 mL) was then added to the resin and allowed to shake at room temperature for 40 min.The resin was then washed with CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL) to afford a light yellow-green resin.Fmoc-Xaa-OH (4 eq.relative to resin loading), PyBOP (4 eq.relative to resin loading) and N-methylmorpholine (8 eq.relative to resin functionalization) in DMF (final concentration 0.125 M of amino acid) was added to the resin, which was shaken at room temperature for 1 h.The resin was then washed with DMF (5 x 3 mL), CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL).The resin was then capped via treatment with 10 vol.% acetic anhydride in pyridine (5 mL) for 5 mins at room temperature.The resin was then washed again with DMF (5 x 3 mL), CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL) prior to determination of the estimated loading of the first amino acid.

Loading to Rink Amide Resin
Rink amide resin (0.6 mmol/g loading) was swollen in dry CH2Cl2 for 30 min, followed by washing with CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL).Fmoc-Xaa-OH, DIC and Oxyma (4.0:4.0:4.4 eq.relative to resin loading, respectively) in DMF (final concentration 0.125 M of amino acid) were added to the resin, which was shaken at room temperature for 1 h.The resin was then washed with DMF (5x 3 mL), CH2Cl2 (5x 3 mL) and DMF (5 x 3 mL).The resin was then capped via treatment with 10 vol.% acetic anhydride in pyridine (5 mL) for 5 mins at room temperature.The resin was then washed again with DMF (5x 3 mL), CH2Cl2 (5 x 3 mL) and DMF (5 x 3 mL) prior to determination of the estimated loading of the first amino acid.

Automated Synthesis of Model Peptides
Peptides were synthesized on a Biotage SYRO I automated peptide synthesizer according to the general setup below.Coupling solutions -Fmoc-protected amino acids (0.

Cleavage from the Resin
Resin-bound peptides were treated with a TFA:iPr3SiH:water mixture (90:5:5 v/v/v, 5 mL) and allowed to shake at room temperature for 2 h.For YPT6 peptides, ammonium iodide (20 eq.) was also added to the cleavage cocktail.At this point, the resin was filtered and washed with fresh cleavage cocktail.The combined cleavage solutions were worked-up as described below.

Work-up and Purification
Ice-cold Et2O (40 mL) was added dropwise to the concentrated cleavage solution to precipitate the crude peptide.The precipitate was then collected via centrifugation and washed further with Et2O to remove any remaining scavengers.Residual Et2O was removed under gentle nitrogen flow and the pellet dissolved in 0.1% v/v TFA aqueous buffer (with minimal MeCN to aid dissolution, if necessary).The crude peptide was analysed by LC-MS (ESI) and purified by RP-HPLC.

General Procedure for PMB Deprotection
The PMB-protected selenopeptide was dissolved in a cocktail of TFA:DMSO:6 M Gn.HCl, 0.1 M HEPES buffer (3:1:1 v/v/v, 7 mg/mL peptide concentration) and the resulting solution was gently agitated at room temperature.Upon complete PMB deprotection (as judged by UPLC-MS), the reaction mixture was diluted with water and purified by RP-HPLC to afford the corresponding peptide diselenide dimer.
General procedure for acyl hydrazide to selenoester conversion: The following describes the general optimized procedure for selenoester formation under which all conversions were conducted unless otherwise stated.
A buffer solution containing 200 mM TCEP, 200 mM HEPES, 50 mM DPDS and 6 M Gnd.HCl was freshly prepared in MilliQ water and the pH was adjusted to 1.5-2.0 using 5 M aqueous HCl, followed by sparging of the solution with argon for 15-20 min.The peptide or protein acyl hydrazide substrate was then dissolved in the buffer (to final concentrations of 250 µM), followed by the addition of 5 eq. of acetylacetone (acac) from a 150 mM aqueous stock.The solution was then allowed to stir at room temperature under an atmosphere of argon for 2-3 h.Completion of the selenoesterification reaction was monitored by UPLC-MS followed by Et2O extraction of residual DPDS.The crude reaction mixture was either subjected directly to a DSL reaction (in the case of the EPSL methodology) or subjected to HPLC purification and lyophilization to afford the purified peptide selenoester as a white fluffy solid.

Ubiquitin acyl hydrazide to selenoester conversion:
Recombinant expression and purification of Ubiquitin-hydrazide: The DNA sequence of human ubiquitin (Ub) was amplified by PCR (primer sequences attached) and subsequently cloned upstream of the Mycobacterium xenopi DNA Gyrase A (Mxe GyrA) intein, a His7 tag and a chitin-binding domain (CBD) into a pTXB1 (New England Biolabs) plasmid via NdeI and SpeI restriction sites.Protein expression was performed using the E. coli BL21(DE3) Gold (Agilent) strain and 2YT medium (16 g/L Trypton, 10 g/L Yeast extract, 5 g/L NaCl) containing 100 µg/mL ampicillin at 37 °C.Overnight cultures were diluted to OD600 0.2, grown until OD600 0.7 and protein overexpression was induced with 1 mM IPTG.After 2 h, the culture was centrifuged for 20 min at 10,000 g, the cell pellets were resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 8) and lysed twice MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPD QQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG in a high-pressure cell disrupter (Constant Systems).The lysate was centrifuged for 30 min at 50,000 g.Each 45 mL supernatant was incubated with 5 mL TBS-equilibrated chitin resin (NEB) for 2 h.The resin was washed twice with 45 mL TBS and subsequently incubated with 30 mL hydrazine solution (5% hydrazine in TBS, 100 mM DTT) for 48 h to produce the Ub-hydrazide.After cleavage, the supernatant was separated from the chitin resin by centrifugation, filtered and purified by RP-HPLC on a C4 column (Kromasil C4, 300 Å, 10 μm, 21.2 mm × 250 mm; 5 to 30% B over 5 min then 30 to 70% B over 40 min, 0.1% v/v TFA, flow rate: 10 mL/min).

General procedure for one-pot selenoesterification, ligation and deselenization (EPSL)
The protein acyl hydrazide substrate was converted to the corresponding selenoester according to the general procedure above.The peptide diselenide dimer fragment was then added to the crude reaction mixture and the pH carefully raised to pH 5 using 5 M NaOH(aq) and the reaction was allowed to proceed at room temperature, with completion of the reaction assessed by UPLC-MS.Following completion of the ligation (typically in 40-50 min), Et2O extraction (x10) was performed to remove residual DPDS.Solid TCEP (40 mM) and reduced glutathione (40 mM) were then added and the pH of resulting solution was adjusted to 7.0 followed by thorough sparging with argon.The reaction mixture was incubated at room temperature or 37 o C for 16 h as specified in the experimental procedure.

II. Synthesis of YPT6 lipopeptides:
A) Synthesis of Alkylated Cysteine Building Blocks
After centrifugation, the supernatant was concentrated in an Amicon Ultra-0.5 device with a 10 kDa molecular weight cut-off and analyzed via RP-HPLC by isocratic elution with 50 mM potassium phosphate buffer at pH 7 containing 10 mM tetrabutylammonium bromide (TBAB) and 8% MeCN.Absorption of nucleotides was followed at 256 nm.
For the folding of dodecylated YPT6 ( 6) at 50 µM initial concentration, no signal for nucleotide bound to the protein after dialysis and concentration was detected.However, analysis of the precipitate collected after dialysis by SDS-PAGE revealed that dodecylated YPT6 ( 6) was precipitated (Figure S16).Higher molecular weight species also indicate irreversible aggregation.Palmitylated YPT6 ( 7) was dissolved at 10 µM initial concentration and no precipitated protein was detected after folding and centrifugation.Analysis of the soluble protein revealed a mixture 29 of GTP and GDP bound to the protein, indicating folding (Figure S17).The ratio of GDP to GTP is increased due to the intrinsic, low GTPase activity of YPT6.

Semi-synthesis of Hsp27 phosphoforms:
Recombinant Expression and Purification of Hsp27(1-172) hydrazide (15): The Hsp27(1-172)-intein fusion protein was expressed from an already available plasmid pTXB3-Hsp27-Mxe-CBD containing the human Hsp27(1-172) DNA sequence followed by the Mxe GyrA intein, a His7 tag and a chitin binding domain. [6]Protein expression was performed in E. coli BL21(DE3) Rosetta 2 grown in 2YT medium (16 g/L Trypton, 10 g/L Yeast extract, 5 g/L NaCl) containing 100 µg/mL ampicillin and 30 µg/mL chloramphenicol at 37 °C.Overnight cultures were diluted to OD600 0.2, grown until OD600 0.7 and protein overexpression was induced by addition of 1 mM IPTG.After 4 h, the culture was centrifuged for 20 min at 10,000 g, the cell pellets were resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.5) and lysed twice in a high-pressure cell disrupter (Constant Systems).The lysate was centrifuged for 30 min at 50,000 g to separate soluble and insoluble fractions.The overexpressed Hsp27 intein fusion construct was isolated from the insoluble fraction (inclusion body) by dissolving the pellet obtained from 2 L culture in 25 mL of solubilization buffer (6 Gnd•HCl, 50 mM Tris, pH 7.5).
After another centrifugation at 50,000 g the clear protein solution was transferred into a Sephadex G-25 PD10 column (Cytiva) and subjected to a buffer exchange into urea buffer (8 M urea in 50 mM Tris.HCl, pH 7.5).Intein cleavage was initiated by diluting the buffer exchanged protein solution 1:1 with 10% hydrazine, 200 mM DTT (final concentration 5% hydrazine, 100 mM DTT, 4 M urea) and incubating at room temperature for 24 h.After cleavage, the Hsp27(1-172) hydrazide 15 was purified by RP-HPLC on a C4 (Kromasil) column (25 to 70% B over 40 min, 0.1% v/v TFA, flow rate: 10 mL/min).(1-172)-NHNH2.This is often observed for proteins overexpressed in bacteria and has no functional consequences]. [6](C) Deconvoluted mass spectrum of 15; The masses correspond to the presence and absence of the Nterminal methionine derived from the expressed Hsp27 segment (vide supra).
After filtering off the resin, the deprotection solution was concentrated under nitrogen flow and the crude peptide was precipitated from ice-cold Et2O.The crude peptide was purified, subjected to PMB deprotection (using standard conditions outlined in the general procedures) and purified by RP-HPLC (0 to 60% B over 55 min, 0.1% v/v TFA) to afford Hsp27 (pSer176) diselenide 18 as a fluffy white solid after lyophilization (6.0 mg, 7% yield).
After filtering off the resin, the deprotection solution was concentrated under nitrogen flow and the crude peptide was precipitated from ice-cold Et2O.The crude peptide was purified, subjected to PMB deprotection (using standard conditions outlined in the general procedures) and purified by RP-HPLC (0 to 60% B over 55 min, 0.1% v/v TFA) to afford Hsp27 (pSer199) diselenide 19 as a fluffy white solid after lyophilization (4.0 mg, 4.5% yield).C) Semi-synthesis of Hsp27 phosphoforms via one-pot selenoesterification, ligation and deselenization:

Figure S22: (A)
1. Hsp27 (pThr174) (12): Hsp27 (pThr174) (12) was synthesized by subjecting Hsp27 (1-172)-NHNH 2 15 (2.0 mg, 0.103 μmol) to the selenoesterification conditions described in the general procedure, followed by the addition of the Hsp27 (173-205, pThr174) diselenide 17 (0.74 mg, 0.206 μmol) in accordance with the general procedure for ligation described above.After Et2O extraction to remove residual DPDS, the reaction mixture was subjected to deselenization as described in the general procedure (60 mM TCEP, 60 mM reduced GSH, pH 6.5, 16 h, room temperature) to afford full length Hsp27 (pThr174) (12) as a fluffy white solid after RP-HPLC purification (0 to 15% B over 5 min then 15 to 75% B over 70 min, 0.1% v/v TFA) and lyophilization (0.95 mg, 40% yield over 3 steps).2. Hsp27 (pSer176) (13): Hsp27 (pThr176) 13 was synthesized by subjecting Hsp27 (1-172)-NHNH 2 15 (2.0 mg, 0.103 μmol) to the selenoesterification conditions described in the general procedure, followed by the addition of the Hsp27 (173-205, pSer176) diselenide 18 (0.74 mg, 0.206 μmol) in accordance with the general procedure for ligation described above.After Et2O extraction to remove residual DPDS, the reaction mixture was subjected to deselenization as described in the general procedure (60 mM TCEP, 60 mM reduced GSH, pH 6.5, 16 h, room temperature) to afford full length Hsp27 (pSer176) (13) as a fluffy white solid after RP-HPLC purification (0 to 15% B over 5 min then 15 to 75% B over 70 min, 0.1% v/v TFA) and lyophilization (1.15 mg, 49% yield over 3 steps).3. Hsp27 (pSer199) (14): Hsp27 (pThr199) 14 was synthesized by subjecting Hsp27 (1-172)-NHNH 2 15 (2.0 mg, 0.103 μmol) to the selenoesterification conditions described in the general procedure, followed by the addition of the Hsp27 (173-205, pSer199) diselenide 19 (0.74 mg, 0.206 μmol) in accordance with the general procedure for ligation described above.After Et2O extraction to remove residual DPDS, the reaction mixture was subjected to deselenization as described in the general procedure (60 mM TCEP, 60 mM reduced GSH, pH 6.5, 16 h, room temperature) to afford full length Hsp27 (pSer199) (14) as a fluffy white solid after RP-HPLC purification (0 to 15% B over 5 min then 15 to 75% B over 70 min, 0.1% v/v TFA) and lyophilization (1.0 mg, 42% yield over 3 steps).Chaperone assay for Hsp27 phosphoforms: The assay was performed according to the literature method with minor modifications. [7]Citrate Synthase (CS) from porcine heart was purchased from Sigma-Aldrich (Taufkirchen, Germany) as an ammonium sulfate suspension then centrifuged to remove most of the ammonium sulfate salts and dialyzed against the storage buffer (50 mM Tris•HCl, 2 mM EDTA, pH 8), final concentration 20-30 µM.The accurate concentration was then determined using bicinchoninic acid (BCA) assay (MW of CS = 48,969 Da) and this stock solution was flash frozen into liquid nitrogen in small aliquots (200-500 µL) and stored at -80 °C.Amorphous aggregation of CS was monitored via measuring the absorbance at 400 nm in a SAFAS UVmc2 double-beam UV-Vis spectrophotometer equipped with a temperature controlled multi-cell holder (SAFAS, Monaco) in 700 µL quartz cuvettes (Hellma Analytics, Germany), 600 µL final volume in triplicate.CS stock solution (obtained as above) was diluted with 40 mM HEPES•KOH (pH 7.5) to a final concentration of 2 µM, and the resulting solution was used as such (control) or treated with Hsp27 variants (0.45 µM final concentration) followed by incubation at 45 °C while measuring the absorbance at 400 nm over 45 minutes (600 µL final volume in triplicate).Prior to the addition, all Hsp27 variants (unmodified recombinant material and synthetic phosphorylated variants as lyophilized powders) were dissolved into 6 M guanidine hydrochloride in 40 mM HEPES•KOH (pH 7.5) buffer to a concentration of approx.1.5 mg/mL, followed by dialysis against 40 mM HEPES•KOH (pH 7.5) buffer, overnight.The accurate concentration of these primary stocks and that of CS were determined via BCA assay and another stock of all Hsp27 samples of concentration 1 mg/mL was prepared.A baseline correction employing only the assay buffer (40 mM HEPES•KOH, pH 7.5) was also performed.The raw data were exported from SAFAS software as a Microsoft Excel worksheet and processed using Microsoft Excel and OriginPro.The results were expressed as average relative UV absorbance at 400 nm, where relative absorption at 400 nm = (absorption at 400 nm)/(maximal absorption at 400 nm by aggregating CS in the absence of a chaperone).

Figure S17 :
Figure S17: RP-HPLC analysis of nucleotides.Left: GTP used in this experiment.Right: Palmitylated YPT6 (7) after folding showing GTP and GDP bound to the protein.