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Introduction to spin label electron paramagnetic resonance spectroscopy of proteins
Author(s) -
Melanson Michelle,
Sood Abha,
Török Fanni,
Török* Marianna
Publication year - 2012
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.20677
Subject(s) - electron paramagnetic resonance , site directed spin labeling , spin label , chemistry , spectroscopy , covalent bond , crystallography , protein structure , nuclear magnetic resonance spectroscopy , deoxygenated hemoglobin , cysteine , analytical chemistry (journal) , nuclear magnetic resonance , hemoglobin , chromatography , stereochemistry , organic chemistry , biochemistry , physics , quantum mechanics , enzyme
An undergraduate laboratory exercise is described to demonstrate the biochemical applications of electron paramagnetic resonance (EPR) spectroscopy. The β93 cysteine residue of hemoglobin is labeled by the covalent binding of 3‐maleimido‐proxyl (5‐MSL) and 2,2,5,5‐tetramethyl‐1‐oxyl‐3‐methyl methanethiosulfonate (MTSL), respectively. The excess spin label is removed by gel‐exclusion chromatography. Changes in the mobility of the reporter groups attached to the protein are monitored by EPR spectroscopy. While the spectral parameters of the rigidly attached 5‐MSL provide information on the rotation of the whole spin labeled protein, MTSL bound by a more flexible linkage describes the local environment of the cysteine residue in the interior of the protein structure. Students can study the known crystal structure of hemoglobin in comparison to the results they obtain by analyzing the EPR spectra. Overall, the exercise introduces them to laboratory techniques such as protein labeling, gel filtration, EPR spectroscopy, as well as familiarizes them with the online Protein Data Bank as a research resource and PyMOL software as a structure visualization tool. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(3):156–162, 2013