Nmr tube

All chemical reagents were of analytical grade. Solvents ethanol, ethyl acetate, and deuterated chloroform (CDCl₃) were purchased from Merck, & Co. (Shanghai, China). NMR tubes were purchased from Wilmad LabGlass (Vinland, NJ, USA). Purified water was obtained by using a Milli-Q system (Millipore, Bedford, MA, USA).

The samples were prepared in an anaerobic chamber and contained: PPPi (1 mM), WT, R190C or E193K ATR (1.1 mM active sites), MgCl₂ (2 mM), AdoCbl (1 mM) in 50 mM Hepes buffer pH 7.5 containing 150 mM KCl and 2 mM DTT at room temperature. The samples were transferred to 5-mm (internal diameter) NMR tubes (Wilmad-Glass), and the final volume was brought up to 0.4 ml with 90% water and 10% D₂O (Cambridge Isotope Labs).
³¹P-decoupled NMR spectra were collected at 242.5 MHz on a Bruker AVANCE NEO spectrometer equipped with a prodigy cryoprobe locked to D₂O. All the NMR experiments were carried out at 25 ^°C using 12 μs 90° pulse widths, 8192 data points, and 4096 scans with 0.8 s recycle time. The chemical shifts were reported on proton-decoupled spectra by Gaussian line fits to the singlet resonances, relative to an external standard of 85% H₃P0₄.

950 MHz Bruker Ascend Aeon and 750 MHz Oxford NMR spectrometers equipped with cryogenically cooled triple-resonance probes were used for all NMR experiments. Spectra were recorded at 25°C in NMR buffer [20 mM Tris pH 7.3, 50 mM NaCl, 1 mM DTT, 0.02%(w/v) NaN₃, 5%(v/v) D₂O]. 2D NMR experiments were carried out using Wilmad NMR tubes (Precision, limit 600 MHz frequency), while Shigemi NMR tubes were used for triple-resonance experiments.

Vortexed
hydrogel solutions are pipetted into NMR tubes (Wilmad-LabGlass, Vineland,
New Jersey) and allowed to cure in a 37 °C oven for 20 min. The
samples are kept at 25 °C until measurements are made. Diffusion
measurements were made at 25, 30, and 37 °C. The samples are
allowed to equilibrate in the instrument (600 MHz Varian NMR spectrometer,
Agilent Technologies, Santa Clara, California) at 25 °C for 10
min and at 30 and 37 °C for 1 h before measurements at each respective
temperature are started. For each temperature, the π/2 pulse
time is determined before making measurements. A stimulated spin-echo
pulse sequence, based on the sequence described by Wu et al., was
used for diffusion coefficient measurements.^{20 (link),21 (link)} The expected signal for this measurement is a function of the Stejskal–Tanner
variable, X, defined in terms of the pulse–sequence
parameters as Experiments used gradient-pulse amplitudes, g, that varied from 0 to 61.5 G/cm with a fixed gradient-pulse
duration (δ = 2 ms) and gradient-pulse separation time (Δ
= 100 ms). The variable-time delay, τ, was computed to ensure
proper timings for a measurement and was typically 1.5 ms. A diffusion
coefficient, D, can then be calculated using where S(X) is the signal for a given value of X and S(0) is the signal without a gradient pulse. The signal
is taken from the water peak in the ¹H NMR spectrum, around
4.0 ppm.