Protein Electrostatics Investigated through Paramagnetic NMR for Nonpolar Groups

Experimental validation of theoretical models for protein electrostatics remains rare. Recently, we have developed a paramagnetic NMR-based method for de novo determination of effective near-surface electrostatic potentials, which allows for straightforward examination of electrostatic models for biomolecules. In the current work, we expand this method and demonstrate that effective near-surface electrostatic potentials can readily be determined from ¹H paramagnetic relaxation enhancement (PRE) data for protein C_αH and CH₃ groups. The experimental data were compared with those predicted from the Poisson-Boltzmann theory. The impact of structural dynamics on the effective near-surface electrostatic potentials was also assessed. The agreement between the experimental and theoretical data was particularly good for methyl ¹H nuclei. Compared to the conventional pK_a-based validation, our paramagnetic NMR-based approach can provide a far larger number of experimental data that can directly be used to examine the validity of theoretical electrostatic models for proteins.