NMR measurements of phase-separating samples

Dear Requester

Below I attach the details of the NMR measurements of phase separating samples as described in the manuscript. These are standard experiments available on all commercial NMR spectrometers (in this case Bruker Avance III) and are very straightforward to set-up. I attach the exact pulse programs that we used that you can apply directly. Please let us know (martin.blackledge@ibs.fr) if any other parts of the protocol are not clear. 

NMR measurements of phase separating samples

¹⁵N labelled P₅₀N₅₂₅ and P_304-507 were premixed at different ratios and diluted in buffer so that final NaCl concentration was 150 mM and then loaded in 3 mm NMR tube. ¹H-¹⁵N HSQCs and R_1r experiments were measured as described for non-phase separating samples. Pulsed field gradient NMR diffusion experiments were measured using ¹⁵N-edited stimulated-echo (STE) diffusion experiments using a 300 ms diffusion delay at 16 to 20 gradient strengths ranging linearly from 0 to 0.55 T m^-1 (47). ¹H-¹⁵N HSQCs used for the development of the kinetic model were measured using both P₅₀N₅₂₅ and P_304-507 ¹⁵N labeled samples at constant P_304-507 concentration of 75 µM and P₅₀N₅₂₅ 5%, 10%, 20% 50% 100%. The concentration of P₅₀N₅₂₅ and P_304-507 in the dilute phase was estimated on the basis of intensities of 23 for P₅₀N₅₂₅ and 9 for P_304-507 non-overlapping and non-interacting peaks which were summed and normalised by the intensities of the peaks in the spectra of the individual proteins. 

Category