2.4. NMR Analysis

Protein samples were prepared in NMR buffer using SEC and centrifugal filters to a final concentration of 0.35–0.5 mM. The samples were transferred to 5 mm D₂O-susceptibility matched Shigemi tubes, and 5 µM sodium 3-(trimethylsilyl)-1-propanesulfonate (DSS) and 10% D₂O were added for chemical shift referencing and locking, respectively. ¹H,¹⁵N-Heteronuclear single quantum coherence (HSQC) spectra were recorded on an Advance III HD 500 MHz instrument (Bruker Corp., Billerica, MA, USA) at 300 K (27 °C) with 32–64 scans and 256 increments. Spectra were processed and analyzed with Bruker Topspin software.

For the chemical shift perturbation analysis, ¹⁵N-Scc4 was concentrated to 0.5 mM and transferred to a 5 mm D₂O-susceptibility matched Shigemi tube. DSS (5 µM) and 10% D₂O were added for chemical shift referencing and locking, respectively. Scc1-FLAG was concentrated to make two stock solutions in NMR buffer: Stock 1 was 3.75 mM and Stock 2 was 7.5 mM. ¹H,¹⁵N-HSCQ spectra were collected on an Advance III HD 500 MHz instrument (Bruker Corp., Billerica, MA, USA) at 300 K (27 °C) with 8 scans and 256 increments. The ¹⁵N-Scc4 sample was analyzed with increasing mole fractions of Scc1-FLAG (0, 0.25, 0.5, 1.0, 1.5, and 2.0 moles of Scc1-FLAG to moles of ¹⁵N-Scc4). Stock 1 was used for the 0.25 and 0.5 mole fraction additions of Scc1-FLAG, and Stock 2 was used to increase the mole fraction to 1.0, 1.5, and 2.0. The sample volume over the titration was 300–350 μL, which is within the detectable region of the NMR probe such that dilution did not affect the signal intensities. After each addition of Scc1-FLAG, the samples were incubated at room temperature for 30 min. For the 1:2 molar ratio (¹⁵N-Scc4:Scc1-FLAG) sample, an additional HSQC spectrum was collected after the sample was incubated overnight at 4 °C. The chemical shift perturbation was calculated by adding the combined chemical shift change [23] using a ¹⁵N shift scaling factor of 0.14 for each step of the titration (since the changes were non-linear).