Analyzing CW EPR Spectra of Nitroxide Labeled Macromolecules

Historically, the primary result of an EPR experiment is the CW EPR spectrum, typically displayed as the first derivative of the absorption spectrum as a function of the magnetic field. Beyond very qualitative assessments, the detailed analysis of an experimental EPR spectrum is a difficult inverse problem. Given a set of parameters and a model, it is easy to calculate a spectrum, but given an EPR spectrum, it is a challenge to decide on the correct model and find all defining parameters of interest. Programs to simulate and fit CW EPR spectra have been around for a long time. Except for a very well-defined model system, an experimental spectrum of a spin labeled protein is typically a mix of multiple states. This article focuses on the analysis of the CW spectrum in several stages of detail, from qualitative to detailed. The use of the EPR lineshape fitting program MultiComponent developed in the Hubbell lab is used to illustrate common approaches to extract information relevant to protein structure, function, dynamics, and thermodynamics.