Incorporation of Multiple β2-Hydroxy Acids into a Protein In Vivo Using an Orthogonal Aminoacyl-tRNA Synthetase

The programmed synthesis of sequence-defined biomaterials whose monomer backbones diverge from those of canonical α-amino acids represents the next frontier in protein and biomaterial evolution. Such next-generation molecules provide otherwise nonexistent opportunities to develop improved biologic therapies, bioremediation tools, and biodegradable plastic-like materials. One monomer family of particular interest for biomaterials includes β-hydroxy acids. Many natural products contain isolated β-hydroxy acid monomers, and polymers of β-hydroxy acids (β-esters) are found in polyhydroxyalkanoate (PHA) polyesters under development as bioplastics and drug encapsulation/delivery systems. Here we report that β2-hydroxy acids possessing both (R) and (S) absolute configuration are substrates for pyrrolysyl-tRNA synthetase (PylRS) enzymes in vitro and that (S)-β2-hydroxy acids are substrates in cellulo. Using the orthogonal MaPylRS/MatRNAPyl synthetase/tRNA pair, in conjunction with wild-type E. coli ribosomes and EF-Tu, we report the cellular synthesis of model proteins containing two (S)-β2-hydroxy acid residues at internal positions. Metadynamics simulations provide a rationale for the observed preference for the (S)-β2-hydroxy acid and provide mechanistic insights that inform future engineering efforts. As far as we know, this finding represents the first example of an orthogonal synthetase that acylates tRNA with a β2-hydroxy acid substrate and the first example of a protein hetero-oligomer containing multiple expanded-backbone monomers produced in cellulo.


A. General cloning protocols
Antibiotics used in this study were supplied at concentrations recommended by addgene.org for selection of E. coli harboring a plasmid of interest (carbenicillin = 100 µg/mL, spectinomycin = 50 µg/mL).

B. General heat shock transformation protocol
All cell strains used in this study (XL1 blue, Top10, DH5α, BL21, C321.ΔΑ.exp) were chemically competent and transformed with plasmids according to the following heat shock transformation protocol.A 100 μL stock of chemically competent cells was thawed on ice for 10 min, after which either 1 μL of 100-200 ng/μL purified plasmid or 5 μL of plasmid generated from site-directed mutagenesis was added to cells on ice and incubated for 30 min.Cells were then subjected to heat shock at 45 o C for 30 s and then placed back on ice for 5 min.After, 900 μL of SOC Outgrowth Medium (NEB, cat # B9020S) was added to heat shocked cells.100 μL of cells transformed with a plasmid harboring resistance to carbenicillin were plated directly on LB-agar supplemented with carbencillin with no recovery step.Plasmids harboring all other antibiotic resistance genes were allowed to recover at 37 o C with shaking at 200 rpm for 1 h.After, 100 μL of recovered cells were plated on a pre-warmed LB-agar + carbenicillin plate and left to grow overnight at 37 o C in a dry air incubator.

C. Plasmid outgrowth and purification from glycerol stocks
For plasmids available stored as glycerol stocks at -80 o C, the following general outgrowth and purification protocol was used.A toothpick stab of DH5α or XL1-blue cells harboring the relevant plasmid was streaked onto an LB-agar plate containing the requisite antibiotic for plasmid selection and grown overnight at 37 o C in a dry air incubator.The following day, 2 individual colonies were picked and used to inoculate a starter culture consisting of 5 mL LB media + antibiotic.This starter culture was grown overnight in a shaking incubator at 37 o C and 200 rpm.
The following day, overnight cultures were pooled and plasmid was purified using a QIAprep® Spin Miniprep Kit (cat# 27104) following the manufacturer's protocol.Purified plasmids were stored at -20 o C.

D. Site-directed mutagenesis for plasmid variants
Plasmids encoding sfGFP or MatRNA Pyl variants were prepared by site-directed mutagenesis using the KLD Enzyme Mix (NEB, Cat #M0554S) per the following manufacturer's reaction protocol.Briefly, 1 μL of a 2 ng/μL stock of a parent plasmid added to 12.5 μL of Q5® Hot Start High-Fidelity DNA Polymerase along with 1.25 μL of 10 μM forward and reverse primers (Supplementary Table 2) designed for site-directed mutagenesis using NEBaseChanger (nebasechanger.neb.com).The reaction mixture was brought up to 25 μL with MilliQ H 2 O and subjected to around-the-horn PCR to generate a linearized dsDNA plasmid harboring the desired mutations.1 μL of around-the-horn PCR product was combined with 1 μL of 10x KLD Enzyme Mix, 5 μL of 2x KLD Reaction Buffer, and 3 μL of MilliQ H 2 O.The reaction mixture was mixed well by pipetting up and down 5 times and then incubated at RT for 5 min.5 μL of the KLD-treated reaction was then transformed into chemically competent XL1 blue cells according to the General Transformation Protocol above.

A. MaPylRS expression plasmid and coding sequence
The previously reported

C. MaFRSA expression plasmid and coding sequence
The previously published 1 plasmid used to express MaFRSA (pET32A-MaFRSA) was obtained from DH5α glycerol stocks.This plasmid encodes the complete sequence of FRSA, an engineered variant of PylRS carrying two mutations in the active site (N166A & V168A) that alter enzyme preference for substrates with ring-substituted phenylalanine sidechains, 2 preceded by an N-terminal GSS-6xHis-SSG sequence to enable immobilized metal affinity chromatography (IMAC) purification.The stop codon is represented as a black asterisk at the C-terminus.

AHDVHEPWSGAGFGLERLLTIREKYSTVKKGGASISYLNGAKIN* D. MaFRSA expression and purification
Expression and purification of MaFRSA followed a protocol that was identical to that employed for the purification of MaPylRS (section IIB).

A. In vitro synthesis of dsDNA encoding MatRNA Pyl
A double stranded DNA template encoding for MatRNA Pyl was synthesized from complementary DNA oligonucleotides (MaPylT-F and MaPylT-R, oligos 1-2, Supplementary Table 1) ordered from IDT.Briefly, each oligo was resuspended in MilliQ H 2 O to a stock concentration of 100 mM.
A 1 μL aliquot of this solution was added to a PCR tube and diluted to a final concentration of 2 mM upon addition of 23 μL MilliQ H 2 O and 25 μL of GoTaq® G2 Master Mix (Promega).Oligos were annealed and extended using the following protocol on a Bio-Rad C1000 Touch Thermal Cycler: 94 °C for 30 s, 30 cycles of 94 °C for 20 s, 53 °C for 30 s and 68 °C for 60 s, and finally 68 °C for 300 s.
After the annealing and extension protocol, the reactions were supplemented with NaOAc (pH 5.2) to a final concentration of 300 mM and washed 1x with 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol.The aqueous layer was washed 2x with chloroform and the dsDNA product was precipitated by addition of ice cold 200-proof ethanol to a final concentration of 71% (v/v).Samples were placed in a dry ice acetone bath for 30 min and pelleted by centrifugation at 213000 x g, 4 o C for 30 min.Liquid was decanted and pellets were washed once in 71% ethanol followed by a second centrifugation.dsDNA pellets were then resuspended in 100 μL MilliQ H 2 O, quantified using a NanoDrop ND-1000 Spectrophotometer and diluted to a final concentration of 500 ng/μL.The final pure dsDNA template has a T7 promoter immediately preceded by a C to increase T7 transcript yield and a 2'-methoxy modification on the penultimate guanosine of the reverse complement strand to reduce non-templated addition of ribonucleotides by T7 RNA polymerase.The reaction mixtures were then extracted once with a 1:1 (v/v) mixture of acidic phenol (pH 4.5)

DNA sequence for
and chloroform and washed twice with chloroform.After extraction, the acylated tRNA was precipitated by adding ethanol to a final concentration of 71% and incubation in a dry ice-acetone bath for 30 min, followed by centrifugation at 21300 x g for 30 min at 4 °C.After carefully aspirating supernatant, tRNA pellets were left to dry for 15 min at room temperature.
Pellets were then resuspended in 2.0 μL of RNAse-free MilliQ H 2 O 1 μL of which was diluted 1:20 in RNAse-free MilliQ H 2 O and transferred to a high recovery mass spec vial for LC-MS analysis.

B. LC-MS analysis of MatRNA Pyl
The tRNA samples from the enzymatic acylation reactions were analyzed by LC-MS as described in Purification and characterization of MatRNA Pyl (Supplementary Information Section VIII, B.).Because the unacylated tRNA peak in each TIC contained tRNA species that could not be enzymatically acylated (primarily tRNAs that lack the 3′-terminal adenosine), 4 simple integration of the acylated and non-acylated peaks in the absorbance at 260 nm (A 260 ) chromatogram could not accurately quantify the acylation yield.To accurately quantify the acylation yield, we used a published procedure. 1,5For each sample, mass data were collected between m/z = 500 and 2,000.A subset of the mass data collected defined as the raw MS deconvolution range was used to produce the deconvoluted mass spectra.The raw MS deconvolution range of each macromolecule species contained multiple peaks corresponding to different charge states of that macromolecule.Within the raw mass spectrum deconvolution range we identified the most abundant charge state peak in the raw mass spectrum of each tRNA species (unacylated, monoacylated and diacylated tRNA).To quantify the relative abundance of each species, the exact mass of the major ions (±0.3000Da) was extracted from the TIC to produce the EICs.The EICs were integrated and the areas of the peaks that aligned with the correct peaks in the TIC (as determined from the deconvoluted mass spectrum) were used to quantify the yields.The expected masses of the oligonucleotide products were calculated using the AAT Bioquest RNA Molecular Weight Calculator, and the molecular masses of the small molecules added to them were calculated using ChemDraw 19.0.

V. Cloning of MatRNA Pyl-opt variants
A. Design of MatRNA Pyl-opt variants pMega-MaPylRS-MatRNA Pyl was purified from a glycerol stock of DH5α cells with using the plasmid outgrowth and purification protocol described in General Cloning Protocols with the use of spectinomycin as the antibiotic selector.
We designed 2 additional pMega-MaPylRS plasmids bearing either MatRNA Pyl-opt1 or MatRNA Pyl-opt2 , chimeric tRNAs with mutations originating from evolved MbtRNA Pyl-opt or EctRNA Sec respectively (Supplementary Figure 6) and synthesized them via site-directed mutagenesis.The following day, cells were harvested via centrifugation in a Beckman Coulter Allegra® X-14R

B. Cloning MatRNA
Benchtop Centrifuge in a Beckman SX4750 Swinging Bucket Rotor at 4300 x g for 1 h.Clarified media was decanted and cell pellets were resuspended in 10 mL Lysis Buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, pH 6.8) + 1 cOmplete ™ , Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche).The cell suspension was passed through at 18 gauge needle to break up cell clumps and cells were lysed via homogenization (Avestin Emulsiflex C3).Briefly, the homogenizer was primed with 50 mL Lysis Buffer after which cell suspension was added and outlet tubing was placed above the inlet cup creating a closed loop.Cells were then passed through the homogenizer at a flow rate of 16.7 mL/min and lysed via homogenization with 15,000-20,000 PSI pulses for 2 min.After homogenization, lysate was collected into a fresh 50 mL conical tube.Lysate was clarified by centrifugation in a Beckman Coulter Allegra® X-14R Benchtop Centrifuge in a FX6100 Fixed-Angle Aluminum Rotor-6 x 100 mL at 11,000 x g for 1 h.During lysate clarification, 1 mL (0.5 mL of packed resin) of TALON ® resin slurry (Takara Bio) was equilibrated into Lysis Buffer by first spinning down resin at 1000 x g for 1 min and decanting storage solution following by 4 subsequent resuspensions and spins in Lysis Buffer.TALON ® resin was added to clarified lysate in a fresh 50 mL conical tube and allowed to equilibrate via batch binding at 4 o C on a rotisserie for 1 hr.Protein was quantified using a NanoDrop ND-1000 Spectrophotometer and concentrated using Amicon Ultra-2 Centrifugal Filter Unit MWCO 10 kDa as necessary for analysis by SDS-PAGE and LC-HRMS.

Preparative scale expression of sfGFP variants in defined media
sfGFP variants expressed in a defined media followed the exact same expression and purification protocols as the above section apart from media formulation.The minimal media recipe was adapted from a published protocol. 8,9See Supplementary Table 2 for components of the defined media used in this study.Agilent, G7120AR).The mobile phases used for separation were 0.1% formic acid in water (mobile phase A) and 100% acetonitrile (mobile phase B), and the flow rate was 0.4 ml min −1 .
After an initial hold at 5% B for 0.5 min, the proteins were eluted using a linear gradient from 5% to 75% B for 9.5 min, a linear gradient from 75% to 100% B for 1 min, a hold at 100% B for 1 min, a linear gradient from 100% to 5% B for 3.5 min and finally a hold at 5% B for 4.5 min.

LC-MS/MS Analysis of sfGFP
Glu-C Digestion.Each sfGFP sample (38 -50 µg total protein) was denatured with 6 M guanidine in a 0.15 M Tris buffer at pH 7.5, followed by disulfide reduction with 8 mM dithiothreitol (DTT) at 37°C for 30 min.The reduced sfGFP sample was alkylated in the presence of 14 mM iodoacetamide at 25°C for 25 min, followed by quenching using 6 mM DTT.
The reduced/alkylated protein was exchanged into 50 µL of 0.1 M Tris buffer at pH 7.5 using a Microcon 10-kDa membrane.To each sample, 6 µg of Glu-C (in a 1.0 µg/µL solution) was added directly to the membrane to achieve an enzyme-to-substrate ratio of at least 1:8 integration in an automated fashion.A preliminary data analysis revealed that Tyr was incorporated into the TAG codon position in most samples.Therefore, Tyr is treated as the "native" amino acid residue at that location, and all other residues are considered as modified form of Tyr.Supplementary Figure 11 shows the possible species incorporated at the TAG position.These species, in addition to the 20 amino acids, were searched by MassAnalyzer.
Peptides containing the three intact monomers usually need to be assigned manually due to the special fragmentation pattern of the Boc group.

VI. Metadynamics simulations of β 2 -hydroxy acid-tRNA in the ribosome
As described in the Results, the starting point for our MD simulations was the RRM as reported in our previous study.The structure was solvated using the simple point charge water model. 10 and Cl-ions corresponding to 0.15 M concentration were added as well as K+ counterions to neutralize the system.The final simulation box measured 100 Å along each side and consisted of ~88,000 atoms.The OPLS4 force field 11 In vitro transcription of MatRNA PylMa-tRNA Pyl was transcribed in vitro using a modified version of a published procedure.3Transcription reactions (25 µL) contained the following components: 40 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 mM DTT, 2 mM spermidine, 5 mM ATP, 5 mM cytidine triphosphate, 5 mM guanosine triphosphate, 5 mM uridine triphosphate, 20 mM guanosine monophosphate, 0.2 mg/mL bovine serum albumin, 20 mM MgCl 2 , 12.5 ng/µL DNA template and 0.025 mg/mL T7 RNA polymerase.The reaction mixtures were incubated at 37 °C in a thermocycler for 3 h.To each 25 µL reaction was added 3.125 U of RQ1 RNAse-free DNAse I (Promega) and 3.125 μL of 10x RQ1 DNAse buffer.Reactions were then incubated at 30 o C for 30 min.After incubation, 8x transcription reactions were pooled (250 μL total) and NaOAc (pH 5.2) was added to a final concentration in 300 μL.The transcription reaction mixtures were then extracted once with a 1:1 (v/v) mixture of acidic phenol (pH 4.5) and chloroform and washed twice with chloroform.To the samples was added ice cold 200-proof ethanol to a final concentration of 71% (v/v) and incubated for 30 min in a dry ice-acetone bath.Samples were spun at 21300 x g for 30 min at 4 o C to pellet RNA, after which liquid was decanted and RNA was dried for 20 min.

C. Expression of sfGFP variants 1 . 2 .
Pyl-opt variants into pMega-MaPylRS vector pMega-MaPylRS plasmids encoding either MatRNA or MatRNA were synthesized by site-directed mutagenesis as described under Site-directed mutagenesis protocol using pMega-MaPylRS-MatRNA Pyl as the parent plasmid.5 μL of the KLD-treated reaction was then transformed into chemically competent XL1-Blue cells as described under General heat shock transformation protocol.After heat shock transformation and recovery, 100μL of recovered cells were plated on a pre-warmed LB-agar + spectinomycin plate and left to grow overnight at 37 o C in a dry air incubator.After overnight growth, 5 individual colonies per construct from overnight growths were picked and used to inoculate 5mL of LB + spectinomycin and grown overnight in a shaking incubator at 200 rpm and 37 o C. Plasmid from liquid cultures was isolated and purified using a QIAprep® Spin Miniprep Kit (cat# 27104) per manufacturer's instructions using a vacuum manifold.Typical yields were in the range of 45 μL of 150-300 ng/μL of pure plasmid.Correct variants were sequence verified by sanger sequencing using the Berkeley DNA Sequencing Core Facility and whole plasmid sequencing from Primordium Labs.VI.Cloning of sfGFP-TAG variantsA.Design of sfGFP-TAG variants pET22b-sfGFP-3TAG was purified from a glycerol stock of DH5α cells by following the same outgrowth and plasmid purification protocol as described above for pET32A-MaFRSA and pET32A-MaPylRS.sfGFP variants into pET22b vector pET22b plasmids carrying sfGFP variants with an in frame amber (TAG) codon were synthesized by site-directed mutagenesis as described under Site-directed mutagenesis protocol using pET22b-sfGFP3TAG as the parent plasmid.5 μL of the KLD-treated reaction was then transformed into chemically competent XL1-Blue cells as described under General heat shock transformation protocol.The pET22b plasmid encodes resistance to carbenicillin so after heatshock and resuspension cells were immediately plated on LB-agar + carbenicillin and left to grow overnight at 37 o C in a dry air incubator.After overnight growth, 5 individual colonies per construct from overnight growths were picked and used to inoculate 5mL of LB + carbenicillin and grown overnight in a shaking incubator at 200 rpm and 37 o C. Plasmid from liquid cultures was isolated and purified using a QIAprep® Spin Miniprep Kit (cat# 27104) per manufacturer's instructions using a vacuum manifold.Typical yields were in the range of 45 μL of 100-200 ng/μL of pure plasmid.Correct sfGFP variants were sequence verified by sanger sequencing using the Berkeley DNA Sequencing Core Facility and whole plasmid sequencing from Primordium Labs.Plate reader-based expression assay of sfGFP variants Chemically competent E. coli cells (C321.ΔΑ.exp,BL21 (DE3), or Top10) were doubly transformed with (1) synthetase plasmid (pMega-MaPylRS or pMega-MaFRSA) and (2) sfGFP reported plasmid (pET22b-sfGFP-3TAG, pET22b-sfGFP-213TAG, pET22b-sfGFP-213-insTAG-214, or pET22b-sfGFP-214TAG) and plated onto selective LB-Agar (100μg/mL carbenicillin + 50μg/mL spectinomycin).The following day, a single colony per double transformant was picked and used to inoculate 5mLs of LB + carb + spec, and grown overnight at 37 o C with shaking at 200 rpm.The following day, 200 μL of overnight culture was used to inoculate 19.8 mL of TB + carb + spec, and grown at 37 o C with shaking at 200 rpm until reaching an OD 600 = ~1.0-1.2 (typically 4.5 -5 h).To 646.75 μL of cell culture at appropriate OD 600 was added IPTG (1 mM final concentration) and monomer (10 mM final concentration).To a Corning® 96-well Flat Clear Bottom Black Polystyrene TC-treated Microplate (Cat# 3904), 200 μL of cell culture + IPTG and monomer was added per well with n = 3 technical replicates per condition.The plate was sealed with a gas permeable Breathe-Easy ® sealing membrane (USA Scientific, Cat # 9123-6100).The plate was incubated at 37 °C for 24 h with continuous shaking in an Agilent Synergy HTX Multi-Mode Plate Reader.Two readings were made at 10 min intervals: (1) the absorbance at 600 nm, to measure cell density, and (2) sfGFP fluorescence with excitation at 485 nm and emission at 528 nm.Preparative scale expression of sfGFP variants pMega-MaPylRS and one pET22b-sfGFP expression plasmid (3TAG, 213TAG, 213TAG214, or 214-TAG) were transformed into C321.ΔA.exp as described in Plate reader-based expression assay of sfGFP variants.From transformants plated on selective LB-agar a single colony was picked and used to inoculate 5 mLs of LB + carb + spec and grown overnight at 37 o C with shaking at 200 rpm.The following day, 1 mL of overnight starter culture was added to 99 mL of liquid TB + carb + spec and monomer 1, 2, 3, or 4 was added to a final concentration of 0.1 mM.Growths were incubated at 37 o C with shaking at 200 rpm until reaching an OD 600 = ~1.0-1.2 and expression of MaPylRS and sfGFP was induced by addition of IPTG (1 mM final concentration).Growths were incubated at 37 o C with shaking at 200 rpm for 18 h.
and Desmond MD system (Schrödinger Release 2023-2) as implemented within Schrödinger Suite (release 2023-2) were used in this study.For all the non-α-amino acid monomers, the Force Field Builder (Schrödinger release 2023-2)11 was used to parametrize the missing torsions.The systems were initially minimized and equilibrated with restraints on all solute heavy atoms, followed by production runs with all but the outer 10 Å C1′ and Cα atoms unrestrained.The constant-temperature, constant-pressure (NPT; number of particles N, pressure P, temperature T) ensemble was used with constant temperature at 300 K and Langevin dynamics.Desmond 12 (Schrödinger release 2023-2) was used for the metadynamics runs.The metadynamics production runs were carried out in duplicate (starting from different conformations as outlined above) for 100 ns each.The Nα-Csp2 distance and the Bürgi-Dunitz angle were used as collective variables.The biasing Gaussian potential ('hill') of 0.01 kcal mol-1 was used, and a width of 0.15 Å for the Nα-Csp2 distance and 2.5°for the Bürgi-Dunitz angle αBD were applied.Analysis of the runs was performed with Schrödinger's Python API (Schrödinger release 2023-2) as well as in-house Python scripts.B.
MaPylRS was used to inoculate a 5 mL starter culture of LB media + 100 μg/mL carbenicillin and grown overnight at 37 o C with shaking at 200 rpm.The following day, 1 mL of starter culture was used to inoculate 100 mL of terrific broth (TB) with 100 μg/mL carbenicillin and grown at 37 o C with shaking at 200 rpm.When cultures reached OD 600 = ~1.0,protein expression was induced by addition of IPTG to 1 mM final concentration and incubated at 18 o C with shaking at 200 rpm for 18 h.The following day, cells were harvested via centrifugation in a 1 plasmid used to express MaPylRS (pET32A-MaPylRS) was obtained from DH5α glycerol stocks.This plasmid encodes the complete sequence of Pyrrolysyl-tRNA synthetase from Methanomethylophilus alvus (Uniprot ID: M9SC49) preceded by an N-terminal GSS-6xHis-SSG sequence to enable immobilized metal ion affinity chromatography (IMAC) purification.Stop codon is represented as a black asterisk at the C-terminus.competent E. coli BL21(DE3) cells (NEB) according to the manufacturer's protocol and plated onto LB-agar plates containing 100 μg/mL carbenicillin.Plates were incubated overnight at 37 o C in a dry air incubator.The following day a single colony of E. coli BL21(DE3) carrying pET32A-

Purification and characterization of MatRNA Pyl
H 2 O according to the manufacturer's protocol.The tRNA was precipitated once more in ice cold 71% ethanol, resuspended in water, quantified using a NanoDrop ND-1000 Spectrophotometer, aliquoted and stored at −20 °C.
To remove small molecules, the tRNA was resuspended in MilliQ H 2 O and further purified using Bio-Rad Micro Bio-Spin™ P-30 Gel Columns, Tris Buffer (RNase-free) after first exchanging the column buffer with MilliQ C.5 µM EDTA (free acid) in 50% MilliQ water-50% methanol (mobile phase B).The analysis was performed at a flow rate of 0.3 ml min −1 and began with mobile phase B at 22%, increasing linearly to 40% B over 10 min, followed by a linear gradient from 40% to 60% B for 1 min, a hold at 60% B for 1 min, a linear gradient from 60% to 22% B over 0.1 min and then a hold at 22% B for 2.
OH 3, about 27 and 80 µg were injected for analysis, respectively, due to the poor signal intensities of these samples.The MS data were collected at 120k resolution setting, followed by data-dependent higher-energy collision dissociation (HCD) MS/MS at a normalized collision energy of 25%.

Table 2 : Recipe for defined Δgln media Reagent Final Concentration Volume for 100 mL of
***2 mg/mL of each amino acid except for Tyr, Cys, and Gln