(2) Rotational correlation time (τc) estimate

Average ¹⁵N relaxation times were determined from 1D ¹⁵N-edited T₁ and T₂ (CPMG) experiments on U-[¹³C, ¹⁵N]-labeled ZF4-5 recorded at a Varian Inova 600 MHz at 298 K. Longitudinal T₁ relaxation delays were 50, 100, 200, 300, 400, 600, 800, 1000, 1500, and 2000 ms; transverse T₂ relaxation CPMG delays were 10, 20, 30, 50, 70, 100, 130, 170, 210, and 250 ms; both experiments had 1.5 s recycle delays. T₁ and T₂ relaxation times were obtained by integration from 8.5 to 10.5 ppm, and τ_c was approximated following the literature equation.⁷ The isotropic overall rotational correlation time of U-[¹³C, ¹⁵N]-labeled ZF4-5 was 7.3 ns based on the backbone ¹⁵N T₁ and T₂ relaxation time measurements. From the linear fit of protein molecular weight (M.W.) versus correlation time for a series of standard proteins,⁸ ZF4-5 was estimated to have a M.W. of 9.6 kDa (Supporting Information Fig. S1). This result indicated that INSM1 ZF4-5 under the NMR study conditions existed predominantly as a monomer (unlabeled ZF4-5 M.W. of 9.2 kDa).