Cysteine Modification 27
local environment can significantly alter the cysteine pKa (Jones et al. 1975 ; Shaked
et al. 1980 ; Houk et al. 1987 ). For example, in cysteine proteases the catalytic thiol
pKa is about 3.5 (Lewis et al. 1978 ; Shipton and Brochlehurst 1978 ). The environ-
mental dependence of the cysteine pKa may effect the reaction rates of sulfhydryl
reagents with engineered cysteines as will be discussed below.
Over the past century, chemists have developed numerous reagents that can co-
valently modify cysteine residues with a high degree of specificity under physi-
ologically relevant experimental conditions. The major families of cysteine modi-
fying reagents include alkyl/aryl halides (i.e., iodoacetamide and iodoacetic acid)
(Dickens 1933 ), maleimides (Smyth et al. 1964 ; Witter and Tuppy 1960 ), mercuri-
als (Boyer 1954 ; Vansteveninck et al. 1965 ), and methanethiosulfonates (Berliner
1983 ; Roberts et al. 1986 ). Thiol modification by the alkyl halides and maleimides
results in formation of stable thioether bonds that cannot by reversed by simple
reducing agents such as 2-mercaptoethanol (2-ME), dithiothreitol (DTT) or Tris(2-
carboxyethyl)phosphine (TCEP) (Thompson and O’Donnell 1961 ; Cleland 1964 ;
Crawhall and Segal 1966 ; Han and Han 1994 ; Singh et al. 1995 ; Getz et al. 1999 ). In
contrast, modification by mercurials, methanethiosulfonates or other reagents that
result in formation of mixed disulfides can, in general, be reversed by the simple
reducing agents. For decades, protein chemists have used these sulfhydryl specific
reagents to investigate the role of endogenous cysteines in the structure and func-
tion of proteins (Vansteveninck et al. 1965 ; Karlin and Bartels 1966 ; Karlin and
Winnik 1968 ).
The ability to clone, mutate and heterologously express proteins greatly ex-
panded the utility of cysteine for exploring protein structure and function. En-
gineered cysteines have been used in several different ways. One approach has
been to insert pairs of cysteine residues to assess the proximity of the chosen
positions by their ability to form disulfide bonds (Careaga and Falke 1992a, b;
Careaga et al. 1995 ; Pakula and Simon 1992 ; Wu and Kaback 1996 ; Yu et al.
1999 ; Horenstein et al. 2001 ). Another major approach has been to insert cyste-
ines either sporadically or systematically. Sporadic cysteine insertion has been
used to establish defined points for site specific protein labeling with fluorescent
and electron paramagnetic probes and other reagents (Berliner 1983 ; Falke and
Koshland 1987 ; Falke et al. 1988 ; Altenbach et al. 1989 , 1990 ; Todd et al. 1989 ;
Jakes et al. 1990 ; Jung et al. 1993 ; Cha et al. 1999 ; Cha and Bezanilla 1997 , 1998 ;
Cha et al. 1998 ; Loots and Isacoff 1998 ; Zheng and Zagotta 2000 ; Chang and
Weiss 2002 ; Blunck et al. 2004 ; Chanda et al. 2004 ; Muroi et al. 2006 ; Pless and
Lynch 2009 ). The use of voltage clamp fluorimetry will be discussed in chapter
Functional Site-Directed Fluorometry and will not be considered further in this
chapter. The era of systematic substitution of cysteine residues was ushered in
with the advent of the Substituted Cysteine Accessibility Method (SCAM) (Aka-
bas et al. 1992 , 1994a, b; Xu and Akabas 1993 ; Karlin and Akabas 1998 ). The
systematic substitution of engineered cysteine residues has been particularly use-
ful for studies of membrane proteins, including ion channels, transporters (Sahin-
Toth and Kaback 1993 ; Yan and Maloney 1993 ; Javitch 1998 ; Chen and Rudnick