Cysteine Modification 33
2.3 Maleimides
Maleimides have an advantage over MTS reagents and mercurials in that they form
a thioether bond that is not readily reversible by commonly used reducing reagents
such as DTT or TCEP (Fig. 1c). This can have significant advantages particularly
for detection of reaction by polyacrylamide gel electrophoresis or where subse-
quent exposure to reducing agents is a potential complication of the experimen-
tal protocol (Karlin and Bartels 1966 ; Falke et al. 1988 ; Sahin-Toth and Kaback
1993 ; Getz et al. 1999 ; Blaustein et al. 2000 ; Lu and Deutsch 2001 ; Skerrett et al.
2001 ). Maleimides react significantly more slowly with thiols in free solution than
mercurials or MTS reagents. The reaction rate of N-ethylmaleimide with cysteine
is ~ 1500 M−1^ s−1 at pH 7 (Gorin et al. 1966 ; Li et al. 2002 ). However, like the
other reagents, maleimides react more rapidly with thiolates than with thiols so
Fig. 2 Crystal structures of
sulfhydryl reagent modi-
fied cysteines. a MTSET-
modified Cys residues from
Streptococcus agalactiae
sortase C1 (PDB # 3TBE).
Protein surface is shown
in dot surface representa-
tion. The MTSET-modified
cysteine is shown in solid
surface representation. Note
that the cysteine sulfur is on
the water-accessible protein
surface. b pCMBS-modified
cysteines from Charcot-
Leyden crystal protein
(galectin-10) (PDB# 1HDK).
The pCMBS-modified cys-
teines are shown in surface
representation, the rest of the
protein is in cartoon represen-
tation. Note that there are two
pCMBS modified cysteines.
Color scheme in both panels,
carbon, teal; sulfur, yellow/
gold; nitrogen, blue; oxygen,
red; hydrogen, green