We have developed a general electron paramagnetic resonance (EPR) method to measure electrostatic potential at spin labels on proteins to millivolt accuracy. Electrostatic potential is fundamental to energy-transducing proteins like myosin, because molecular energy storage and retrieval is primarily electrostatic. Quantitative analysis of protein electrostatics demands a site-specific spectroscopic method sensitive to millivolt changes. Previous electrostatic potential studies on macromolecules fell short in sensitivity, accuracy and/or specificity. Our approach uses fast-relaxing charged and neutral paramagnetic relaxation agents (PRAs) to increase nitroxide spin label relaxation rate solely through collisional spin exchange. These PRAs were calibrated in experiments on small nitroxides of known structure and charge to account for differences in their relaxation efficiency. Nitroxide longitudinal (R1) and transverse (R2) relaxation rates were separated by applying lineshape analysis to progressive saturation spectra. The ratio of measured R1 increases for each pair of charged and neutral PRAs measures the shift in local PRA concentration due to electrostatic potential. Voltage at the spin label is then calculated using the Boltzmann equation. Measured voltages for two small charged nitroxides agree with Debye-Hückel calculations. Voltage for spin-labeled myosin fragment S1 also agrees with calculation based on the pK shift of the reacted cysteine.
Bibliographical noteFunding Information:
This work has been supported from grants to DDT from NIH (AR32961, GM27906). We are grateful to Karol Subczynski and Justyna Widomska in the National Biological EPR Center in Milwaukee (NIH center Grant # EB001980) for assistance in R 1 measurements using saturation recovery. We thank Igor V. Negrashov for excellent programming assistance. We thank Yuri Nesmelov and Adam Burr for helpful discussions. Small molecule modeling was done in Fujitsu Cache ™ .
- Paramagnetic relaxation
- Transition metal complex