Avc 600 mhz nmr spectrometer

NMR samples of pKID-Min4 were prepared with a final peptide concentration of 800 μM, in a solution containing 100 μM TSP (3-trimethylsilyl-2,2,3,3-d₄-propionic acid) and 5 mM NaCl in 90% H₂O/10% D₂O in a total volume of 400 μL. The pH was adjusted to pH 6.5. NMR experiments were conducted in the absence of metal and in the presence of one equivalent of the diamagnetic metal LaCl₃, in order to examine the changes caused by the formation of the metal-phosphopeptide complex. NMR spectra were recorded on a Brüker AVC 600 MHz NMR spectrometer equipped with a triple resonance cryoprobe. TOCSY and NOESY NMR experiments were conducted for sequential residue assignment and for specific resonance assignments. ¹H-¹³C HSQC spectra for the apo peptide were recorded under identical conditions. ¹H-¹³C HSQC spectra for the peptide complex with La³⁺ were recorded using solutions in 100% D₂O. Experiments were conducted using La³⁺ due to the paramagnetism of Tb³⁺ (6 unpaired electrons), which results in severe broadening and peak shifts, precluding detailed analysis of the structure of the peptide using the metal employed for optimization.