Gadolinium-Based NMR Spin Relaxation Measurements of Near-Surface Electrostatic Potentials of Biomolecules

NMR spectroscopy is an important tool for the measurement of the electrostatic properties of biomolecules. To this point, paramagnetic relaxation enhancements (PREs) of H-1 nuclei arising from nitroxide cosolutes in biomolecular solutions have been used to measure effective near-surface electrostatic potentials (phi(ENS)) of proteins and nucleic acids. Here, we present a gadolinium (Gd)-based NMR method, exploiting Gd chelates with different net charges, for measuring phi(ENS) values and demonstrate its utility through applications to a number of biomolecular systems. The use of Gd-based cosolutes offers several advantages over nitroxides for phi(ENS) measurements. First, unlike nitroxide compounds, Gd chelates enable electrostatic potential measurements on oxidation-sensitive proteins that require reducing agents. Second, the large electron spin quantum number of Gd (7/2) results in notably larger PREs for Gd chelates when used at the same concentrations as nitroxide radicals. Thus, it is possible to measure phi(ENS) values exclusively from + and - charged compounds even for highly charged biomolecules, avoiding the use of neutral cosolutes that, as we further establish here, limits the accuracy of the measured electrostatic potentials. In addition, the smaller concentrations of cosolutes required minimize potential binding to sites on macromolecules. Fourth, the closer proximity of the paramagnetic center and charged groups within Gd chelates, in comparison to the corresponding nitroxide compounds, enables more accurate predictions of phi(ENS) potentials for cross-validation of the experimental results. The Gd-based method described here, thus, broadens the applicability of studies of biomolecular electrostatics using solution NMR spectroscopy.