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INSTITUTE OF PHYSICS PUBLISHING JOURNAL OF PHYSICS: CONDENSED MATTER J. Phys.: Condens. Matter 17 (2005) S1459–S1469 doi:10.1088/0953-8984/17/18/004 Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron–electron resonance) of force activation in muscle Piotr G Fajer Institute of Molecular Biophysics, Department of Biological Science, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, USA E-mail: [email protected] Received 9 December 2004 Published 22 April 2005 Online at Abstract The recent development of site speci±c spin labelling and advances in pulsed electron paramagnetic resonance (EPR) have established spin labelling as a viable structural biology technique. Speci±c protein sites or whole domains can be selectively targeted for spin labelling by cysteine mutagenesis. The secondary structure of the proteins is determined from the trends in EPR signals of labels attached to consecutive residues. Solvent accessibility or label mobility display periodicities along the labelled polypeptide chain that are characteristic of β -strands (periodicity of 2 residues) or α -helices (3.6 residues). Low-resolution 3D structure of proteins is determined from the distance restraints. Two spin labels placed within 60–70 Å of each other create alocal dipolar ±eld experienced by the other spin labels. The strength of this ±eld is related to the interspin distance, r 3 .T h e dipolar ±eld can be measured by the broadening of the EPR lines for the short distances (8–20 Å) or for the longer distances (17–70 Å) by the pulsed EPR methods, double electron–electron resonance (DEER) and double quantum coherence (DQC). Abrief review of the methodology and its applications to the multisubunit muscle protein troponin is presented below. (Some ±gures in this article are in colour only in the electronic version) 1. Introduction The last decade witnessed the rapid developmentofstructural methods resulting in a nearly exponential increase of solved protein structures. Attention is now shifting from structure determination as a goal in itself towards a description of the structural changes that facilitate the function of biological macromolecules. This new goal has created an increasing need for methods that extend and complement the high resolution static structures of isolated proteins 0953-8984/05/181459+11$30.00 © 2005 IOP Publishing Ltd Printed in the UK S1459
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S1460 PGFajer in crystals to descriptions of proteins in solution and in large macromolecular complexes that are functionally active. Furthermore, the increasing realization of the signi±cance of structural heterogeneity and the order-to-disorde rt ransitions in many biological processes gives a premium to methods that are sensitive to structural heterogeneity and disorder. Equally important is the addition of timeresolution to structural methods so that protein dynamics and/or structural transitions accompanying biological activity are captured.
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