Abstract
Investigations on the structure and function of biomolecules often depend on the availability of topological information 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.
Highlights
Understanding the function of biomacromolecules requires information about reaction kinetics, structure and conformational dynamics
Structures of biomolecules and their complexes are determined by means of X-ray crystallography [10] or high-resolution NMR spectroscopy [8]
Crystallization might be difficult or even impossible, especially for membrane proteins, and this method usually fails where the function relies on the flexibility of the structure or weak interactions between two molecules, recently X-ray techniques became available which allow studying the structural dynamics of proteins in solution [7]
Summary
Understanding the function of biomacromolecules requires information about reaction kinetics, structure and conformational dynamics. Böhme et al / Site-directed spin labeling and DEER spectroscopy suited for accessing distances in the nanometer range, dealing with complex structures and high molecular weights as well as providing information on flexible, less ordered structures [1,11,26] Within this class of methods EPR has two major advantages over fluorescence techniques. For nitroxides distance distributions rather than mean distances, to which FRET is limited, can be determined On ground of these benefits, during the past years site-directed spin labeling (SDSL) in combination with EPR spectroscopy has developed as an efficient tool to access the nanometer length scale and to elucidate structure and conformational dynamics of biomolecules. This article briefly summarizes the basics of distance measurements by EPR on spin-labeled biomacromolecules, with special emphasis on its application to the tRNA modifying enzyme MnmE
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