Pfg onenmr probe

Between 300–700 μM of each Hsp33^M172S-tFPA variant was incubated in 40 mM KPi, 5 mM DTT, supplemented with 2 mM trifluoracetic acid for intensity and chemical shift referencing, and 10% (v/v) D₂O. All ¹⁹F NMR experiments were performed on a 500 MHz Varian VNMRS spectrometer (Agilent Technologies), equipped with a 5 mm PFG OneNMR probe. ¹⁹F spectra were acquired using a single 90° pulse with carbon decoupling. ¹⁹F temperature-dependent spectra were acquired between 10 and 45 °C in steps of 5 °C. Peptide titration spectra of Hsp33 with NPY/NPY^D4C-IAM-TEMPO were performed at 30 or 35 °C. Data were processed with iNMR.

Thawed 400 μL urine samples will be mixed with 200 μL of a 0.75 M phosphate buffer solution (containing 0.5 % sodium trimethylsilylpropionate-d4 (TSP)) prepared in D₂O and transferred to a 5 mm NMR tube. Thawed 200 μL blood serum samples will be mixed with 400 μL D₂O and transferred to a 5 mm NMR tube. All ¹H NMR spectra will be collected on a Varian 500 MHz NMR spectrometer (Agilent technologies, Santa Clara, United States) equipped with a 5 mm PFG One NMR probe. One dimensional(1D) NOESY-Presaturation pulse sequence for urine and 1D Carr-Purcell Meiboom-Gill (cpmg) experiment for blood serum will be used. Each free induction decay (FID) will be collected using a total of 128 scans with a relaxation delay of 2 s.