Non-Linear Continuous Wave Electron Paramagnetic Resonance Spectroscopy for Investigation of Lipid-Protein...

This chapter describes how to utilize spin-spin interactions measured by electron paramagnetic resonance (EPR) to gain structural details of lipid-protein interactions in samples containing more than one type of paramagnetic molecules. Common in these methods is that certain types of continuous wave (CW) EPR signals (also called spectral displays) are recorded under partial microwave saturation, extending into the region of a non-linear dependence of some spectral features on the microwave power (hence, the name non-linear CW EPR), and spectral parameters are derived that are proportional to the longitudinal (T₁) relaxation time, which is in turn highly sensitive to spin-spin interaction between the paramagnetic molecules. Although powerful pulsed EPR techniques exist to measure T₁ (and also for the transversal relaxation time, T₂) directly, the advantage of the methods described here is that they can be performed on the much cheaper and more widespread conventional CW EPR instruments. During the 1990s, we developed and refined T₁-sensitive nonlinear CW EPR techniques, which were then combined with paramagnetic quenching agents and doubly spin-labeled samples for distance measurements. In the following sections, first the concept of non-linear CW EPR will be introduced, then the common materials and methods will be described, followed by four example protocols and data analysis, and the chapter is concluded with useful notes. This chapter is incremental to the previous one in the sense that several experimental details are the same, such as the sources and types of spin-labeled lipid analogues, preparation of lipid-labeled bio- and model membranes, assaying lipid and protein content, sample geometry, basic instrument settings and processing of the EPR spectra (e.g., removal of the background and disturbing spectral components, normalization). Here, the focus is on the extra experimental and theoretical procedures required for measuring various types of spin-spin interactions and utilizing them for proximity relations in membranes. The example procedures and figures are illustrations, rather than exact reproductions of data from our previous works.