Avance 850 mhz nmr

¹⁵N-labeled proteins were expressed in M9 minimal media containing ¹⁵NH₄Cl and glucose. Purified ¹⁵N-labeled SERF1a was concentrated to 0.1 mM in 50 mM Tris-HCl (pH 6.8), 20 mM NaCl, and 3 mM NaN₃ for NMR structural studies. ¹⁵N-SERF1a alone as reference was 70 μM and titrated with TrxHttex1-39Q at the indicated ratio. All NMR experiments were carried out at 298 K on Bruker Avance 850 MHz NMR or 600 MHz spectrometers equipped with 5 mm triple resonance cryoprobe and Z-gradient. Data were acquired and processed using the software Topspin2.1 (Bruker, Germany) and further analyzed using Sparky version 3.114 (Goddard and Kneller). 1H chemical shifts were externally referenced to 0 ppm of 2,2-dimethyl-2-silapentane-5-sulfonate, and ¹³C and ¹⁵N chemical shifts were indirectly referenced in accordance with IUPAC recommendations^{50 (link)}. Protein backbone resonance assignments were based on standard triple resonance experiments^{51 (link)}: HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HNCA, and HN(CO)CA. The chemical shift perturbation for combined ¹H and ¹⁵N resonances of SERF1a was calculated using the following equation: Δppm = [(5*Δ¹H)² + (Δ¹⁵N)²]^{1/2 52 (link)}. The intensity drop rate was normalized to K62, which shown the least intensity drop, that is, I (bound)/ I (free) of K62 was assumed to be 1.00.

¹⁵N-labeled proteins were expressed in M9 minimal media containing ¹⁵NH₄Cl and glucose. Purified ¹⁵N-labeled SERF1a was concentrated to 0.1 mM in 50 mM Tris-HCl (pH 6.8), 20 mM NaCl, and 3 mM NaN₃ for NMR structural studies. ¹⁵N-SERF1a alone as reference was 70 μM and titrated with TrxHttex1-39Q at the indicated ratio. All NMR experiments were carried out at 298 K on Bruker Avance 850 MHz NMR or 600 MHz spectrometers equipped with 5 mm triple resonance cryoprobe and Z-gradient. Data were acquired and processed using the software Topspin2.1 (Bruker, Germany) and further analyzed using Sparky version 3.114 (Goddard and Kneller). 1H chemical shifts were externally referenced to 0 ppm of 2,2-dimethyl-2-silapentane-5-sulfonate, and ¹³C and ¹⁵N chemical shifts were indirectly referenced in accordance with IUPAC recommendations^{50 (link)}. Protein backbone resonance assignments were based on standard triple resonance experiments^{51 (link)}: HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HNCA, and HN(CO)CA. The chemical shift perturbation for combined ¹H and ¹⁵N resonances of SERF1a was calculated using the following equation: Δppm = [(5*Δ¹H)² + (Δ¹⁵N)²]^{1/2 52 (link)}. The intensity drop rate was normalized to K62, which shown the least intensity drop, that is, I (bound)/ I (free) of K62 was assumed to be 1.00.

¹⁵N-labeled proteins were expressed in M9 minimal media containing ¹⁵NH₄Cl and glucose. Purified ¹⁵N-labeled SERF1a was concentrated to 0.1 mM in 50 mM Tris-HCl (pH 6.8), 20 mM NaCl, and 3 mM NaN₃ for NMR structural studies. ¹⁵N-SERF1a alone as reference was 70 μM and titrated with TrxHttex1-39Q at the indicated ratio. All NMR experiments were carried out at 298 K on Bruker Avance 850 MHz NMR or 600 MHz spectrometers equipped with 5 mm triple resonance cryoprobe and Z-gradient. Data were acquired and processed using the software Topspin2.1 (Bruker, Germany) and further analyzed using Sparky version 3.114 (Goddard and Kneller). 1H chemical shifts were externally referenced to 0 ppm of 2,2-dimethyl-2-silapentane-5-sulfonate, and ¹³C and ¹⁵N chemical shifts were indirectly referenced in accordance with IUPAC recommendations^{50 (link)}. Protein backbone resonance assignments were based on standard triple resonance experiments^{51 (link)}: HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HNCA, and HN(CO)CA. The chemical shift perturbation for combined ¹H and ¹⁵N resonances of SERF1a was calculated using the following equation: Δppm = [(5*Δ¹H)² + (Δ¹⁵N)²]^{1/2 52 (link)}. The intensity drop rate was normalized to K62, which shown the least intensity drop, that is, I (bound)/ I (free) of K62 was assumed to be 1.00.