bdSUMO-HSPB611 (link)−20 (link) proteins in 25 mM Tris pH 7.4, 25 mM NaCl at 5 mM concentration
were diluted 10-fold in a buffer with pH ranging from 4 to 10. This
dilution buffer contained 100 mM sodium acetate (pKa 4.8), 100 mM Bis-Tris (pKa 6.5), 100 mM Tris (pKa 8.1), 100 mM
CHES (pKa 9.3), 25 mM NaCl, and 10% D2O, and its pH was between 4 and 10. By including four reagents
with pKa’s ranging from 4.8 to
9.3, buffering was effective from pH 4 to 10. Thus, all proteins for
NMR analysis, regardless of pH, were in the identical buffering matrix.
After dilution, any insoluble protein was precipitated by centrifugation.
Samples were then transferred to 5 mm NMR tubes (Norell) and experiments
were conducted on a Bruker Advance III 500 MHz NMR Spectrometer outfitted
with a 5 mm BBOF probe. Data were collected with a spectral window
of 99.5774 ppm, 65,536 real plus imaginary points, a D1 of 2.0 s,
and between 1,024 and 13,000 scans. All NMR experiments were collected
at 25 °C. NMR data were processed (apodized, zero filled, Fourier
transformed, and phased) and analyzed in Bruker Topspin. Labeled peaks
were exported to Graphpad Prism 8 and plotted against the pH of the
buffer; the plotted points were interpolated to a Sigmoidal, 4 PL
function, and the pKa2 was determined
at the calculated inflection point ±95% Confidence Interval.
were diluted 10-fold in a buffer with pH ranging from 4 to 10. This
dilution buffer contained 100 mM sodium acetate (pKa 4.8), 100 mM Bis-Tris (pKa 6.5), 100 mM Tris (pKa 8.1), 100 mM
CHES (pKa 9.3), 25 mM NaCl, and 10% D2O, and its pH was between 4 and 10. By including four reagents
with pKa’s ranging from 4.8 to
9.3, buffering was effective from pH 4 to 10. Thus, all proteins for
NMR analysis, regardless of pH, were in the identical buffering matrix.
After dilution, any insoluble protein was precipitated by centrifugation.
Samples were then transferred to 5 mm NMR tubes (Norell) and experiments
were conducted on a Bruker Advance III 500 MHz NMR Spectrometer outfitted
with a 5 mm BBOF probe. Data were collected with a spectral window
of 99.5774 ppm, 65,536 real plus imaginary points, a D1 of 2.0 s,
and between 1,024 and 13,000 scans. All NMR experiments were collected
at 25 °C. NMR data were processed (apodized, zero filled, Fourier
transformed, and phased) and analyzed in Bruker Topspin. Labeled peaks
were exported to Graphpad Prism 8 and plotted against the pH of the
buffer; the plotted points were interpolated to a Sigmoidal, 4 PL
function, and the pKa2 was determined
at the calculated inflection point ±95% Confidence Interval.