Chapter 20
Electron Paramagnetic Resonance Techniques for Measuring Distances in Proteins
Downloaded by TUFTS UNIV on December 6, 2016 | http://pubs.acs.org Publication Date: August 7, 2002 | doi: 10.1021/bk-2002-0827.ch020
Sandra S. Eaton and Gareth R. Eaton Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80208
Analysis of the effects of electron-electron dipolar interactions on electron paramagnetic resonance (EPR) spectra of samples with interacting paramagnetic centers can be used to determine the distance between a pair of interacting spins. The technique required to measure the dipolar interaction depends upon the distance between the spins and on the electron spin relaxation times. Continuous wave spectra can be analyzed to obtain distances up to about 20 Å, and longer distances can be measured by pulsed methods. Changes in relaxation times can be used to determine the distance between a slowly relaxing spin and a more rapidly relaxing spin.
Deciphering Biological Function and Structure at a Molecular Level A central goal of science is to explain biological function at a molecular level, which therefore requires knowledge of the structure of the biological system. As genome research rapidly increases the number of proteins known, the requirement for obtaining structural information increases dramatically (7). © 2002 American Chemical Society Eaton et al.; Structures and Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 2002.
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Downloaded by TUFTS UNIV on December 6, 2016 | http://pubs.acs.org Publication Date: August 7, 2002 | doi: 10.1021/bk-2002-0827.ch020
322 Estimates of distances between parts of proteins or assemblies of proteins with other species can be made by exploiting diffraction, energy transfer, and dipolar interactions. The most common of these methods have been X-ray diffraction, fluorescence energy transfer, NMR, and EPR. Each physical method provides a different perspective, and multiple methods are needed to achieve a frill view (2). For each method, key questions include for what species is it appropriate, what range of distances can be measured, how sensitive is the method to distributions in distance or in orientation, and what is the accuracy and precision of the method. Furthermore, one wants to know whether a method is sensitive to dynamics, and whether this sensitivity blurs the distance measurement or is an opportunity to measure dynamics of distances. X-ray diffraction can yield high-resolution interatomic distances for the form of the protein that is stable in a crystal. When the crystal is cryocooled the resolution for a protein may be as good as 1.2 Â. However, two things that X-ray diffraction does not reveal are (a) the structure of the protein in solution or other physiological environment, and (b) the electronic configuration of a metal in a metalloprotein (2). Even in the small number of cases in which crystals have been formed of proteins that function in a membrane (ca. 20 so far), some parts have remained ill-defined by the single-crystal X-ray diffraction. Thus, membrane-bound proteins are a significant challenge and opportunity for other methods of structure determination. A renaissance in electron microscopy, especially using freeze-trapped conformations and image-processing is providing structures with 20 Â resolution, and in some cases near 3 À resolution (3). Single particles of large complexes can be studied, and when atomic-resolution structures of component parts are available from other techniques, such as X-ray diffraction or NMR, it is possible to put detailed structural information into functional context (3). Many short distances (
