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Spectroscopy 24 (2010) 283–288 283 DOI 10.3233/SPE-2010-0428 IOS Press Accessing the distance range of interest in biomolecules: Site-directed spin labeling and DEER spectroscopy Sabine Böhme, Heinz-Jürgen Steinhoff and Johann P. Klare ∗ Department of Physics, University of Osnabrück, Osnabrück, Germany Abstract. Investigations on the structure and function of biomolecules often depend on the availability of topological informa- tion to build up structural models or to characterize conformational changes during function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site – directed spin labeling (SDSL) allow to determine intra- and intermolecular distances in the range from 4–70 Å, covering the range of interest for biomolecules. The approach does not require crystalline samples and is well suited also for molecules exhibiting intrinsic flexibility. This article is intended to give an overview on pulsed EPR in conjunction with SDSL to study protein interactions as well as conformational changes, exemplified on the tRNA modifying enzyme MnmE. Keywords: Site-directed spin labeling, SDSL, double electron–electron resonance, DEER, distance determination, MTSSL, spin label, MnmE 1. Introduction Understanding the function of biomacromolecules requires information about reaction kinetics, struc- ture and conformational dynamics. The structures and therefore also conformational changes associated with biological function are situated in the Ångström to nanometer range, rendering this length scale most important for biomolecular research. Typically, structures of biomolecules and their complexes are determined by means of X-ray crystal- lography [10] or high-resolution NMR spectroscopy [8]. Both techniques provide information on the atomistic level, but suffer from serious limitations. Crystallization of the biomolecule implies that