Crystalline Spin-Labeled Hemoglobin as a Model to Compare Distances Measured by DEER Spectroscopy and X-Ray Crystallography

Abstract

We have recently crystallized and resolved the structure of spin-labeled hemoglobin, while simultaneously using double electron-electron resonance (DEER) spectroscopy to measure inter-spin-label distances within these crystals. Previously, no spin-labeled protein has been analyzed by both DEER-spectroscopy and X-ray crystallography to determine whether the two techniques are in good agreement. Human hemoglobin (Hb) is a useful model system for this comparison, as it readily crystallizes and reacts specifically with the maleimide-TEMPO spin-label (MSL) at Cys93, found within the β subunits of the α2β2 hemoglobin tetramer. For our experiments, we have generated two crystal populations. The first consisted entirely of paramagnetic, EPR-active MSL-Hb, which was used in our X-ray crystallography experiments. The second population contained a low concentration of MSL-Hb in a large excess of hemoglobin labeled with an EPR-silent MSL analog, which assured that distances measured by DEER spectroscopy would not be altered by dipolar interactions between spin-labels of adjacent Hb tetramers. Our results show that both techniques yield similar inter-spin distance measurements, provided that certain precautions are taken to avoid EPR artifacts. We have varied DEER acquisition parameters, such as the dipolar evolution time and acquisition temperature, and analysis methods that affect the accuracy and precision of distance distributions observed by DEER spectroscopy, as compared to those obtained by X-ray crystallography. These results provide the most rigorous analysis to date of the reliability of EPR-based distance measurements. This work was supported by NIH grants (GM27906, AR32961, AG26160, RR22362, GM08700).