Cysteine Modification 39
the electrostatic potential in the binding site. They measured the effect of reagent
charge and ionic strength on the reaction rates. The calculated electrostatic potential
in the binding site at zero ionic strength was − 80 mV relative to bulk solution. This
corresponds to 2–3 negative changes in and around the binding site (Stauffer and
Karlin 1994 ). A similar approach was used to measure the electrostatic potential in
the channel lumen (Wilson et al. 2000 ).
Extensive SCAM studies identified residues lining the agonist and allosteric ben-
zodiazepine binding sites in the GABAA receptor (Boileau et al. 1998 , 1999 , 2002 ;
Kucken et al. 2000 ; Teissere and Czajkowski 2001 ). In almost all cases the residues
aligned with the sulfhydryl reactive positions are found in the crystal structures of
Acetylcholine Binding Protein (AChBP) and glutamate binding site of the homolo-
gous glutamate-gated chloride channel (GluCl) (Brejc et al. 2001 ; Celie et al. 2004 ;
Hibbs and Gouaux 2011 ). Thus, SCAM studies correctly identified the residues
lining the agonist and allosteric binding sites. In a variant of SCAM, a sulfhydryl
reactive GABAA receptor agonist, 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione,
protected a number of engineered cysteines from covalent modification. Interest-
ingly, it reacted at a measurable rate with only one binding site engineered cyste-
ines. Presumably the binding site held the thiol reactive moiety in close proximity
to this cysteine. This provided evidence for the relative orientation of this reagent in
the binding site (Jansen et al. 2008 ). Studies with unnatural amino acids, discussed
more extensively in chapter Incorporation of Non-Canonical Amino Acids, have
provided additional insight into the role of aromatic residues in cation-π interac-
tions in the agonist binding sites of Cys-loop receptors (Kearney et al. 1996 ; Beene
et al. 2002 , 2004 ; Lummis et al. 2005 , 2011 , 2012 ; Pless et al. 2008 , 2011 ).
Fig. 4 Crystal structure of the cytoplasmic third of the GluCl channel (PDB# 3RI5) showing pic-
rotoxin bound at the level of the 2’ threonine residues. One subunit has been removed. Protein is
shown in cartoon representation except for the 2’ threonines that are shown in solid surface repre-
sentation. Adjacent subunits have different colors and the threonines are colors by their respective
subunit. Picrotoxin is shown in solid surface representation, carbon, gray; oxygen, red