Glycylglycine

Methanococcus maripaludis strain S2 was obtained from our laboratory collection (Whitman et al.) [27 (link)] and cultured at 37°C. Methanothermococcus okinawensis strain IH1 was obtained from Takai et al. and cultured at 62°C [28 (link)].
Cultures were grown in H₂/CO₂ medium (McNA, a minimal medium with 10 mM sodium acetate) or formate medium (McF) reduced with 3 mM cysteine hydrochloride. The 5 mL cultures were grown in 28 mL aluminum-sealed tubes. For McNA, the tubes were pressurized to 276 kPa with H₂/CO₂ (4 : 1, v/v) and refilled with the same gas every 24 hours after inoculation. Detailed protocols for growth on formate are given in Appendix A. Briefly, McF medium contained 0.4 M sodium formate and was buffered with 0.2 M glycylglycine (pH = 8.0). The medium was first sparged with N₂ to remove most of the O₂, and 3 mM cysteine chloride was then added. Tubes were pressurized to 103 kPa with N₂/CO₂ (4 : 1, v/v) before autoclaving. Prior to inoculation, 3 mM sodium sulfide was added as the sulfur source.
The buffers tested were obtained from Sigma Chemical Co. and included (with the counter ion) Tricine/NaOH (N-[Tris(hydroxymethyl)methyl]glycine), Bicine/NaOH (N,N-bis(2-hydroxyethyl)glycine), Tris/HCl (2-amino-2-hydroxymethyl-propane-1,3-diol), glycine/NaOH, and glycylglycine/NaOH. During formate medium preparation, ingredients were added as listed in the appendices, and the organic buffers were added from stock solutions at pH 7. The concentration of NaCl was adjusted depending upon the amount of sodium formate and sodium in the buffer used so that the final concentration of sodium ion was 0.4 M.
The final medium was also tested for plating (Appendix B) and growth of 1.5 L cultures (Appendix C).

The induction times of glycine
homopeptides crystallization were determined in water at two different
temperatures, 278.15 K and 283.15 K. Solutions with different supersaturation
levels (listed in Table S2) were prepared
for each glycine homopeptide in test tubes. For glycine and diglycine,
2 mL solutions were made with 2 g of deionized water, whereas a volume
of 1.5 mL was preferred for triglycine due to the increased feasibility
of nucleation. The tubes were equipped with a small magnetic stir
bar and meticulously sealed with rubber lids wrapped with parafilm
both inside and outside the cap. The samples were placed into the
hot thermostat bath maintained at 333.15 K, well above the supersaturation
temperature of glycine homopeptides. The solutions were stirred at
500 rpm in the water bath until all the glycine homopeptides had fully
dissolved (Figure S1). The clear tubes
were then immersed in a cold thermostat bath held at a low constant
nucleation temperature (278.15 K and 283.15 K). During the experiment,
the solutions of different supersaturations for each peptide were
tested in parallel and continuously magnetically stirred at 250 rpm.
The induction time was recorded as the time of the first observation
of the solution becoming cloudy. Once the solutions had nucleated,
the tubes were transferred back to the water bath held at a temperature
above the supersaturation temperature, to dissolve before repeating
the nucleation experiment cycle. Induction time data were obtained
for 100 times for each glycine homopeptide and each supersaturation
level to capture the stochastic nature of nucleation. After all the
induction time measurements were performed, the solutions were filtered,
and the powders were tested using PXRD to determine the morphology
of the crystallized glycine homopeptides to make sure there was no
polymorphism transformation during the nucleation process.

One day before transfection, ∼2.7 × 10⁶ HEK293T cells were seeded in a 10 cm dish using full DMEM. Cells were transfected with 1 µg of PTH1R-encoding plasmid with or without serine substitutions under control of a CMV promoter, 5 µg of the reporter construct pGL4.29 (humanized PpyRE9 firefly luciferase gene driven by a cAMP-responsive element and followed by a PEST-sequence (Branchini et al., 2010), 0.5 µg of plasmid pRL encoding Renilla luciferase driven by a CMV promoter and 3.5 µg of the empty pcDNA3.1 vector. Transfection was performed using PEI as described above. The next day, cells were trypsinized and transferred to poly-d-lysine (PDL) coated 96-well plates with a density of ∼70,000 cells per well. After 24 h, cells were stimulated at 37 °C for 3 h by the addition of 25 µl of PTH(1-34) dissolved in pure DMEM to final concentrations from 10⁻¹² to 10⁻⁶M in a 96-well. Each concentration was analyzed in four wells. The following steps were performed according to Seidel et al. 2017. Cells were washed with ice-cold HDB (12.5 mM HEPES pH 7.4, 140 mM NaCl, 5 mM KCl). Cell lysis was performed using 50 µl of luciferase buffer (10 mM MgSO₄, 25 mM glycylglycine, 4 mM EGTA, pH 7.8) supplemented with 1% Triton X-100 and 1 mM dithiothreitol (DTT) for 30 min on ice under constant gentle agitation. Luciferase activities were measured using a Omega luminometer (BMG LABTECH, Ortenberg, DE) equipped with two injectors. To each well, 50 µl of luciferin substrate buffer (luciferase buffer supplemented with 0.3 mM luciferin, 1 mM ATP, 1 mM DTT pH 7.8) were subsequently added by the first injector and the total luminescence was measured. Afterward, 50 µl of 5 µM colenterazine dissolved in HDB were added to each well (1.67 µM final concentration of coelenterazine in the well). The luminesence of Renilla luciferase was detected using a 475–30 nm emission filter. Firefly luminescence was normalized to the Renilla luminescence. Curves were fitted by non-linear regression using Prism 9 for Windows (Graphpad Software Inc., San Diego, CA). EC₅₀ values were obtained as means with the appropriate CI from at least three independent experiments, each performed in quadruplicate.