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N
N
O
O
O
CH
2
S
CH
2
SH
O
O
O
+
N
N
O
N
N
O
O
O
O
O
H
3
N
H
3
N
MAL-6
N
N
O
O
CH
2
S
O
CH
2
SH
O
+
H
2
C
S
CH
2
O
H
3
C
S
S
N
O
O
N
O
O
H
3
N
O
MTSSL
H
3
N
Figure15.3
Labeling chemistry of the piperidine maleimide spin label (MAL-6) and MTSSL with cysteine side
chains. The MTSL moiety can be removed with DTT or mercaptoethanol to yield the thiol nitroxides and free
cysteine. The molecular volume of the MTSL moiety is similar to that of the tryptophan side chain. (Reprinted
withpermissionfrom[12].Copyright1998PlenumPress.)
15.4.2 The reporter group approach: who makes the news?
While NMR spectroscopists are “purists” in that they observe natural spins (nuclei) without altering the
physical or chemical environment, spin labels and fluorescent probes are sometimes bulky structures that
are introduced exogenously. As previously mentioned, spin labeling was continuously accused of perturbing
the system by “making the news” instead of reporting the news.
3
For some years even the fluorescence
community accused the spin labelers, while neglecting the typically very bulky bi- and tricyclic aromatic
molecules needed in luminescence labeling studies. When substituting a nitroxide side chain at a specific
position in a protein, a minor structural perturbation might occur, but evidence has continually shown that
the majority of these labeling studies, including where they were placed at internal positions in a protein,
did not result in altering structure/function changes, although the thermal stability of the protein may be
altered slightly. The fact is that it is necessary to slightly perturb or interact with the macromolecular
environment in order to report meaningful information.
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