Measurement of ¹H^α transverse relaxation rates in proteins: application to solvent PREs

It has recently been demonstrated that accurate near surface electrostatic potentials can be calculated for proteins from solvent paramagnetic relaxation enhancements (PREs) of amide protons measured using spin labels of similar structures but different charges (Yu et al. in Proc Natl Acad Sci 118(25):e2104020118, 2021). Here we develop methodology for extending such measurements to intrinsically disordered proteins at neutral pH where amide spectra are of very poor quality. Under these conditions it is shown that accurate PRE values can be measured using the haCONHA experiment that has been modified for recording ¹H^α transverse relaxation rates. The optimal pulse scheme includes a spin-lock relaxation element for suppression of homonuclear scalar coupled evolution for all ¹H^α protons, except those derived from Ser and Thr residues, and minimizes the radiation damping field from water magnetization that would otherwise increase measured relaxation rates. The robustness of the experiment is verified by developing a second approach using a band selective adiabatic decoupling scheme for suppression of scalar coupling modulations during ¹H^α relaxation and showing that the measured PRE values from the two methods are in excellent agreement. The near surface electrostatic potential of a 103-residue construct comprising the C-terminal intrinsically disordered region of the RNA-binding protein CAPRIN1 is obtained at pH 5.5 using both ¹H^N and ¹H^α-based relaxation rates, and at pH 7.4 where only ¹H^α rates can be quantified, with very good agreement between potentials obtained under all experimental conditions.