Spin Labeling: A Modern Perspective - Stable Radicals: Fundamentals and Applied Aspects of Odd-Electron Compounds - page 530

Figure15.6 StructureoftheN6-SL-NAD -GAPDHtetrameras determinedfrommolecularmodeling.Allfour

N6-SL-NAD'cofactorsareshowninbold.ThelowerpairofGAPDsubunitsshowsthebackboneonly.Theupper
pair shows the backbone and side chains. The crystallographic R axis extends upward through the tetramer.
(Adaptedfrom[17],Copyright1997,withpermissionfromElsevier.)

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15.6 Site directed spin labeling (SDSL): how is it done?

For site directed spin labeling it is necessary to be able to spin label a protein with “distance markers”
that can analyze aspects of conformation and polarity in the cases of both singly (and multiply) labeled
proteins. Briefly, one (or occasionally two) thiol group(s) must be incorporated in a protein sequence and
subsequently be covalently labeled with a uniquely specific reactive, reversible spin label. As mentioned
earlier, Berliner and coworkers introduced the MTSL label and demonstrated its efficacy with the thiol
protease, papain. 11 This was followed by the groundbreaking work of Hubbell and coworkers who recog-
nized that it was possible potentially to genetically engineer cysteine residues into proteins at will. 13 The
technique is now relatively standard with a series of protocols and parameters for any protein system. The
following, taken in part from Feix and Klug, 12

summarizes the SDSL paradigm and the information that

can be learned from it.