Avance 2 800 mhz spectrometer

In contrast to the more stable FKBP12, at pH 6 and 25°C the ¹H–¹⁵N two-dimensional correlation spectra of FKBP12.6 accumulate additional cross-peaks with a limited dispersion in ¹H chemical shift, as is typical of disordered structures. When the pH is raised to 7 in the presence of thiol reducing agents, samples of the wild-type FKBP12.6 were suitably stable for the time periods needed for multidimensional resonance-assignment experiments. The cysteine-free variant of FKBP12.6 was used for the zz-exchange measurements at elevated temperatures. NMR assignment data were collected on a Bruker Avance III 600 MHz spectrometer and a Bruker Avance II 800 MHz spectrometer at 25°C. Backbone resonance assignments were carried out using standard HNCO (Kay et al., 1994 ▶ ), HN(CA)CO (Kay et al., 1994 ▶ ), HNCACB (Muhandiram & Kay, 1994 ▶ ) and HN(CO)CACB (Yamazaki et al., 1994 ▶ ) experiments. The FELIX software (Felix NMR) was used for NMR data processing. The backbone resonance assignments for the unligated wild-type human FKBP12.6 have been deposited in the Biological Magnetic Resonance Bank (BMRB accession Nos. 19323 and 19324 for the major and minor slow-exchange states, respectively).

Spectra were acquired on an Avance II 800 MHz spectrometer (Bruker) using a 5-mm TCI CryoProbe (Bruker). Data were processed using TopSpin 3.1 (Bruker) and analyzed using CcpNmr analysis software (41 (link)). Samples of ¹⁵N-FH 8–9 and ¹⁵N-PspCN were used to optimize conditions by varying NaCl from 0 to 150 mM (pH from 4 to 6.5) and temperature from 15 to 70°C. Subsequently, 40 μM ¹⁵N-FH 8–9 in 20 mM potassium phosphate (pH 6.2) was used to record a ¹H,¹⁵N HSQC spectrum at 310 K, then PspCN was added to 100 μM and the spectrum was rerecorded. Separately, 30 μM ¹⁵N-PspCN in 20 mM potassium phosphate (pH 6.0) was used to record a ¹H,¹⁵N HSQC spectrum at 298 K, then FH 8–9 was added to 40 μM and the spectrum was rerecorded.

The [¹⁵N]‐ or [¹⁵N, ¹³C]‐labeled PACSIN2 SH3 domain was purified with the same procedures. Backbone assignments were performed with ¹H‐¹⁵N Heteronuclear single quantum coherence (HSQC) recorded at 298 K on a Bruker AVANCE II 600 MHz spectrometer (Gachon University, Incheon). All ¹H‐¹⁵N HSQC NMR titrations were performed at 298 K on a Bruker AVANCE II 800 MHz spectrometer (Korea Basic Science Institute, O‐Chang). For Cobll1‐NT and SH3PB1‐CT titrations, unlabeled Cobll1‐NT and SH3BP1‐CT proteins were purified and prepared in the same buffer (50 mM sodium phosphate, 100 mM NaCl, 1 mM TCEP, pH 6.3). The proteins were titrated to 50 μM of ¹⁵N‐labeled PACSIN2 SH3 domain at various molar ratios (PACSIN2 SH3 domain: Cobll1‐NT:SH3BP1‐CT = 1:0:0, 1:0:1, 1:1:0, 1:1:1). All spectra were processed using NMRpipe and visualized using NMRViewJ software.