Toledo ph probe

Freeze-dried ubiquitin and diubiquitin samples were dissolved in 5% DMSO (Biosolve) in Milli-Q® water and then redissolved in NMR buffer containing 20 mM NaPO₄ pH 6.8 and 10% D₂O. Then, samples were taken in 15 ml 3.5 kDa Millipore spin filter tubes and spun-washed with three volumes of NMR buffer until DMSO was almost completely removed (LC/MS analysis). Concentrated samples were diluted to 500 μL with NMR buffer and the final concentration was determined using BCA assay using ubiquitin as standard. The pH was carefully measured using a Mettler TOLEDO pH probe.
All NMR studies were carried out on a Bruker 900 MHz spectrometer with a TCI cryoprobe, at 298 K (25°C). [¹H, ¹⁵N] HSQC-spectra were acquired, processed and calibrated using standard methods. Chemical Shift Perturbations (CSPs) were calculated by comparing the [¹H, ¹⁵N] HSQC spectra of mono Ub with that of each of the diUb molecules/ The CSP was calculated according to the following formula
where ΔδH and ΔδN are the chemical shift differences for ¹H and ¹⁵N, respectively.
The spectra of K6 diUb indicated two different co-existing conformations. An “open conformation” was assigned based on similarity with the mono-Ub spectrum.

L. plantarum strains B1.1 and B1.3 were previously isolated from Ethiopian teff injera (20 (link)) and were maintained as frozen glycerol stocks at −80°C. L. plantarum was routinely grown at 30°C in a commercial preparation of MRS (cMRS) (BD, Franklin Lakes, NJ), a modified MRS (26 (link)) (mMRS) prepared as lacking beef extract, mMRS supplemented with beef extract (8 g/L) (mMRS-BE), or a teff flour medium. To prepare the teff flour medium, whole-grain teff flour (Bob’s Red Mill, Milwaukie, OR) was sterilized by autoclaving for 1 h and then drying for approximately 15 h at room temperature. Teff flour medium was prepared by mixing the teff flour with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄·7H₂O, 1.4 mM KH₂PO₄) (pH 7.2) at a dough yield [100 × weight (flour + PBS)/weight (flour)] (50 (link)) of 300. pH values were measured after 72 h of incubation in mMRS or mMRS with 5 mM CaCl₂ using a Mettler Toledo pH probe (Mettler Toledo, Greifensee, Switzerland).