DETERMINATION OF THE DISTANCE BETWEEN 2 SPIN LABELS ATTACHED TO A MACROMOLECULE

An EPR ''spectroscopic ruler'' was developed using a series of alpha-helical polypeptides, each modified with two nitroxide spin labels. The EPR line broadening due to electron-electron dipolar interactions in the frozen state was determined using the Fourier deconvolution method. These dipolar spectra were then used to estimate the distances between the two nitroxides separated by 8-25 Angstrom. Results agreed well with a simple alpha-helical model. The standard deviation from the model system was 0.9 Angstrom in the range of 8-25 Angstrom. This technique is applicable to complex systems such as membrane receptors and channels, which are difficult to access with high-resolution NMR or x-ray crystallography, and is expected to be particularly useful for systems for which optical methods are hampered by the presence of light-interfering membranes or chromophores.