title: Introduction to spin label electron paramagnetic resonance spectroscopy of proteins.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Melanson
authors.forename: Abha
authors.initials: A
authors.lastname: Sood
authors.forename: Fanni
authors.initials: F
authors.lastname: Török
authors.forename: Marianna
authors.initials: M
authors.lastname: Török
mesh_terms: D001665:Binding Sites; D003479:Curriculum; D003497:Cyclic N-Oxides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D013334:Students
chemical_list: D003497:Cyclic N-Oxides; D006454:Hemoglobins; D013113:Spin Labels; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide